Immunoablative therapies

ABSTRACT

This invention pertains to pharmaceutical compositions comprising a glucocorticoid for use in the treatment of diseases by immunoablation. The compositions of the invention may be for use in the treatment of diseases that are mediated by immune cells such as lymphocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of European PatentApplication No. 18198491.5, filed Oct. 3, 2018, which is incorporated byreference herein in its entirety for any purpose.

FIELD OF THE INVENTION

This invention pertains to compositions for use in the treatment ofdiseases by immunoablation. In particular, the compositions of theinvention may be for use in the treatment of diseases that are mediatedby immune cells such as lymphocytes.

BACKGROUND OF THE INVENTION

The present inventors had previously found that high concentrations ofglucocorticoids could be used to condition patients to enhance theefficacy of cellular immunotherapies such as adoptive T cell therapy;described in currently unpublished International patent applicationPCT/US2018/025517 (filed 30 Mar. 2018). In that application, theinventors had noted the toxicities associated with chemotherapy andradiation mediated preconditioning, which is believed to non-selectivelydestroy the cellularity of the spleen. The inventors had providedglucocorticoids (a subclass of steroids) and other non-toxiclymphodepleting agents, at acute doses, to benefit cancer patients whoreceive cellular immunotherapies.

International patent application PCT/US2018/025517 notes that high doseglucocorticoids can cause ablation of lymphoid tissues to reduce thebinding of cellular immunotherapies to lymphoid tissue, in particular togerminal centers and marginal zones in lymph nodes and germinal centersand marginal zones in the spleen. International patent applicationPCT/US2018/025517 further notes that the high dose glucocorticoids alsolymphodeplete peripheral blood lymphocytes via a biologic mechanism (incontrast to the cytotoxic mechanism underpinning preconditioning withchemotherapeutic agents or radiotherapy).

Prior studies into the use of steroids to precondition a patient priorto ACT had shown this approach to be ineffective. Hinrichs (JImmunother. 2005 November-December; 28(6):517-24) had evaluateddexamethasone as a preconditioning treatment prior to ACT. In comparisonto total body irradiation (TBI), Hinrichs demonstrated that an HED of0.8 mg/kg administered on day −6, day −4, and day −2 lymphodepletedequivalently to 5Gy TBI. Hinrichs demonstrate that pretreatment withsystemic intraperitoneal dexamethasone at 10 mg/kg (HED 0.81 mg/kg) onday −6, −4, and −2 before ACT induced equivalent lymphodepletioncompared to radiation, but this pretreatment did not enhance ACT tumorkilling. In contrast, Hinrichs discloses that pretreatment withradiation did enhance ACT tumor killing. In the Hinrichs paper, thedexamethasone reportedly caused splenic lymphodepletion as demonstratedby 99% reduced spleen cellularity. However, while Hinrichs reported 99%lymphodepletion, no enhancement of ACT tumor killing was observed. Incontrast, Hinrichs observed that radiation does enhance ACT tumorkilling. Experiments to repeat Hinrichs reported lymphodepletion,however, demonstrate that the Hinrichs doses of intraperitonealdexamethasone at 10 mg/kg (HED 0.81 mg/kg) on day −6, day −4, and day−2, do not effectively lymphodeplete peripheral blood lymphocytes. WithHinrichs dosing, only B lymphocytes in the peripheral blood weresignificantly lymphodepleted, from 10680 (vehicle control) to 3733 liveevents measured by flow cytometry of CD3−CD19+ cells, a 65% reduction.In contrast, CD3+T lymphocytes were reduced from 3370 to 2441 liveevents, only a non-significant 33% reduction. CD3+CD4+T lymphocytes werereduced from 1779 to 902 live events, only a non-significant 50%reduction. CD3+CD8_T lymphocytes were reduced from 1318 to 1277 liveevents, only a non-significant 3% reduction. CD3+CD4+CD25+FoxP3+Tregswere reduced from 198 to 70 live events, only a non-significant 65%reduction. And natural killer (NK) cells were reduced from 1153 to 958live events, only a non-significant 17% reduction.

Autoimmunity is the phenomena of the immune system aberrantly mountingan attack on a subject's own constituents. (In healthy subjects, theimmune system avoids damaging autoimmune reactions by establishingtolerance to the subject's own constituents.) Diseases that result fromdamaging autoimmune reactions are termed autoimmune diseases. Differentautoimmune diseases affect different parts of the body; these can bedebilitating (e.g. in the case of rheumatoid arthritis, which affectsthe joints) neurodegenerative/neurodestructive (e.g. in the case ofmultiple sclerosis) and are in some cases, such as diabetes mellitus,associated with substantial mortality rates (Thomas et al., 2010).

The pathogenesis of autoimmune disorders is widely attributed to acrucial role to T and B lymphocytes inappropriately recognizing selfantigens and initiating a cell-mediated or humoral reaction, or both,resulting in inflammatory tissue and vascular damage (Sullivan et al,2010; Shlomchik et al., 2001).

Autoimmune diseases are very often treated by prolonged administrationof immunosuppressives such as steroids. For instance, pemphigus patientshave been treated with 100 mgs dexamethasone by 2 hour IV infusion dailyfor 3 days (Pasricha et al., 2008). This dose is not lymphoablating. Thepemphigus patients were treated in this way every 28 days until cure. Ittook between 3 and 12 months to cure them. The relapse rate was 15% andall patients went in to remission with another Dexa treatment. This doseof Dexa is between about 1-2 mg/kg. While helping to manage theautoimmune disease and reducing symptoms, such treatment regimens arenot curative, involve several long term side effects and the increasedrisk of infection (Patt et al., 2013).

Lymphodepletion therapies are increasingly tested for controlling immunedamage. One appealing premise for such a therapy is that it may ‘reboot’the immune system and restore immune tolerance (Lu et al., 2011).However, the tolerogenic potential of lymphodepletion therapies remainscontroversial. The debate is exemplified by conflicting evidence fromthe studies of anti-thymocyte globulin (ATG), a prototype ofimmunodepleting drugs, in particular on whether it inducesCD4+CD25+Foxp3+ regulatory T (Treg) cells (Lu et al., 2011). Tounderstand the impact of ATG on T cells at a clonal level in vivo, Lu etal studied the effect of anti-mouse thymocyte globulin (mATG) in areductionist model in which the T-lymphocyte repertoire consists of asingle clone of pathogenic T effector (Teff) cells specific to aphysiological self-antigen. The mATG treatment led to peripheralinduction of antigen-specific Treg cells from an otherwise monoclonalTeff repertoire, independent of thymic involvement. The de novoinduction of Treg cells occurred consistently in local draining lymphnodes, and persistence of induced Treg cells in blood correlated withlong-term protection from autoimmune destruction. (Lu et al., 2011) thusprovides in vivo evidence for clonal conversion from a pathogenicself-antigen-specific Teff cell to a Treg cell in the setting ofimmunodepletion therapies.

Type 1 diabetes mellitus (T1D) is an autoimmune disease thatprogressively results in the depletion of insulin-secreting (3-cellsthat eventually culminates in clinically significant hyperglycemia andmetabolic instability (Atkinson et al., 2014). Overall, T1D accounts forapproximately 5% of diabetes and affects about 20 million individualsworldwide (Menke et al., 2013). About 1.25 million Americans have T1Dand an estimated 40,000 people will be newly diagnosed each year in theU.S (American Diabetes Association, Diabetes Care 37, 2014). TD1 isassociated with an annual economic burden in U.S. of $14.4 billion,considering medical expenses and indirect costs such as lost income.

Therapeutic insulin and other treatment based on external hypoglycemicagents do not cure T1D, but simply offer solutions to control glucoselevel in blood. Patients remain susceptible to labile blood glucoselevels and the development of microvascular and macrovascular diabeticcomplications (Peng et al., 2018).

Safe interventions to remove autoimmune substrates from diabetespatients are missing. Autoimmunity in TD1 includes many arms of theimmune response (Snarski et al 2016; Cantu-Rodriguez et al., 2016). As aconsequence, antigen-specific immunotherapies based on the use ofantibodies, fusion proteins, cytokines, regulatory T cells, andsmall-molecule inhibitors lead only to some degrees of β-cellspreservation and reduction of blood glucose level in patients with T1D(Kim et al., 2013). Even in combinations, immunotherapies targetingspecific components of autoimmunity repertoire failed to guaranteerestoration of insulin independence (Bone et al., 2017).

Autologous hematopoietic stem cell transplantation (HSCT) is so far theonly proven strategy for T1D cure (Voltarelli et al., 2007). AutologousHSCT has been performed for twelve years as a therapeutic option forautoimmune diseases (ADs) such as multiple sclerosis, systemicsclerosis, rheumatoid arthritis, systemic lupus erythematous, Crohn'sdisease and others (Swart et al., 2017). This more intense and widerimmunologic approach consists in an “immunologic reset” performed withhigh-dose immunosuppression which comprises non-specific abrogation ofautoreactive T- and B-cell responses followed by hematopoietic stem celltransplantation for reconstitution of a tolerant immune system.Remarkably, in clinical trials, this approach has enabled up to 80% ofT1D patients to experience periods of insulin independence in parallelwith relevant increments in C-peptide levels during mixed meal tolerancetest (Couri et al., 2018). However, serious concerns are preventing theadoption of immunologic reset as a therapeutic approach for T1D.

Risks associated with the HSCT procedure exceed the positive effectsoffered for T1D: HSCT is still associated with significant toxicitiesand up to 3% mortality (Alexander et al., 2018; Pallera et al., 2004;Henig et al., 2014). Moreover, current protocols for immunologic resetare based on cytotoxic immunosuppressive regimens (e.g. chemotherapy,radiotherapy) that expose patients to a series of safety issuesincluding short-term risks of infection, acute organ dysfunction anddeath, and long-term risks of malignancies and secondary autoimmunediseases (Daikeler et al., 2012).

Almost all T1D patients treated with HSCT resumed exogenous insulin use,with a subsequent decrease in C peptide levels (Magdalena et al., 2018)as the effect of incomplete ablation of autoimmune pathophysiologicsubstrates after preconditioning (PC) (Loh et al., 2007). Increasing theintensity of transplant conditioning regimens or repeating the procedureto improve treatment outcomes would expose patients to excessive risksand toxicities (Couri et al., 2018).

HSCT is associated with high costs, which range from approximately$80,000 to $300,000, depending on conditioning regimens given beforeHSCT, transplant type, and inpatient costs associated withhospitalization (Broder et al., 2017).

A need exists for further treatments of autoimmune disorders and otherdiseases that are mediated by lymphocytes. Further treatments that aresimpler and less costly than HSCT would be desired.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that high dosesof glucocorticoids can act to cause lymphodepletion of peripheral bloodlymphocytes without substantially affecting the cell count of othercells. Further actions such as the ablation of germinal centers alsounderpins certain aspects of this invention. The present inventionprovides medical applications of these actions of high doseglucocorticoid agonists; for use in the treatment of lymphocyte mediateddiseases.

Accordingly, in a first aspect, the invention provides a pharmaceuticalcomposition comprising a glucocorticoid, for use in the treatment of alymphocyte mediated disease in a subject, wherein the treatmentcomprises administering a dose of the pharmaceutical composition to thepatient to deliver the glucocorticoid at a dose equivalent to about 15to about 26 mg/kg human equivalent dose (HED) of dexamethasone base. Thedose of glucocorticoid may be termed an ‘acute high dose’. Thepharmaceutical composition may (or may not) comprise a pharmaceuticallyacceptable carrier as defined herein. The pharmaceutical composition may(or may not) comprise a pharmaceutically acceptable preservative asdefined herein. The pharmaceutical composition may (or may not) comprisea pharmaceutically acceptable chelating agent as defined herein.However, in all embodiments of this aspect, the pharmaceuticalcomposition does comprise one or more ingredients selected from thegroup consisting of: a pharmaceutically acceptable carrier, apreservative, and/or a chelating agent. The pharmaceutical compositionmay also include excipients, in some embodiments. In some embodiments,the pharmaceutical composition comprises more than one pharmaceuticallyacceptable carriers. In some embodiments, the pharmaceutical compositioncomprises more than one pharmaceutically acceptable preservatives. Insome embodiments, the pharmaceutical composition comprises more than onepharmaceutically acceptable chelating agents. Embodiments of thisinvention can be defined as acting to achieve systemic lymphodepletionin the subject. Lymphodepletion involves the removal of B and T cells.

In some embodiments, the lymphocyte mediated disease is an autoimmunedisease, for instance an autoimmune disease selected from the groupconsisting of Type 1 diabetes, multiple sclerosis, amyotrophic lateralsclerosis, scleroderma, pemphigus and lupus. The lymphodepletive actionof the invention underpins the efficacy of these embodiments.

Despite glucocorticoids having a well-established use in many autoimmuneconditions (Flammer et al., 2011) they have never been considered forimmunologic reset. In addition, studies based on the use ofpharmaceutical low doses to precondition patients prior to autologouscell transplant showed this approach to be ineffective (MedicinesAgency; 2017). The complex mode of action based on multiple in vivoeffects of the pharmaceutical composition of the present inventionprovides the first effective replacement of chemotherapy that can beused as safe immunologic reset regimen for treatment of autoimmuneconditions such as diabetes mellitus.

Several advantages are associated with the present invention, relatingto the actions of the pharmaceutical composition, including (i)Non-myeloablative immunologic reset: The pharmaceutical composition candeplete all peripheral blood lymphocyte types, for example, includingislet-specific autoreactive T-cells responsible for diabetesautoimmunity, but spare neutrophils, platelets, RBCs and stem cells(both HSCs and MSCs) based on the specific receptor-mediated mode ofaction. The invention therefore reduces risks of infection and removesthe need of HSCT to recover blood cells after immune-reset. The resultis a non-myeloablative regimen that can perform a safe immunologic resetwith efficacy comparable to chemotherapy. (ii) Reduction of germinalcenters (GCs) and marginal zones in secondary lymphatics. Thepharmaceutical composition transiently ablates germinal centers in thesecondary lymphoid organs that give rise to high-affinity antibodies andlong-lived plasma cells (DeFranco et al., 2016) for increased efficacyover autoimmune pathophysiologic substrates. (iii) Simple modes ofadministration. The pharmaceutical composition can be formulated fororal or intravenous administration routes, making it effective withinhours with lymphocyte and GC recovery within 7-14 days. For the firsttime, complete lymphodepletion will not require hospitalization. (iv)Reduced chances of relapse. Unlike chemotherapy or radiation, thepharmaceutical composition of the invention can be safely administeredat completely lymphoablating doses to remove memory T and B cellresponsible of relapse. (v) Acceptable re-administration. In case ofrelapse of autoimmune pathophysiologic substrates, the safety profile ofhigh dose glucocorticoids will allow repetitive dosing of thepharmaceutical compositions of the invention.

These actions and advantages associated with the present invention,disclosed herein, mean that the skilled person will understand that theinvention provides an effective strategy for treating autoimmunediseases as well as the other lymphocyte mediated diseases discussedherein.

In some embodiments, the lymphocyte mediated disease is residual HIVdisease. In these embodiments, as described herein, a reduced number ofgerminal centers in the subject's lymphoid organs can force residual HIVinfected T cells, which bind to niches in these centers, into thecirculation where they can be eliminated by the immune system orstandard therapies. Within the context of this disclosure, the skilledperson will understand that HIV is a lymphocyte mediated disease in thesense that the virus infects T lymphocytes. The lymphodepletive actionof the invention also contributes to the efficacy of these embodiments.

In other embodiments, the lymphocyte mediated disease is a lymphoma,e.g. a germinal centre lymphoma (GC lymphoma) or marginal zone lymphoma.In these embodiments, as described herein, a reduced number of germinalcenters in the subject's lymphoid organs can force cancer cells (forexample germinal center lymphomas), which bind to niches in thesecenters, into the circulation where they can be eliminated by the immunesystem or standard therapies. The lymphodepletive action of theinvention also contributes to the efficacy of these embodiments. Inparticular embodiments, treatment of Burkitt's Lymphoma (BL) isspecifically envisaged. In Africa, BL treatment revolves around acombination of three chemotherapy drugs, Cyclophosphamide, Vincristine,and Methotrexate (systemic and intrathecal). This combination isrepeated at 2-week intervals for a total of six cycles over 12 weeks(Burkitt's Lymphoma National Treatment Guidelines. 2009). Lower doses ofdexamethasone are currently on the WHO List of Essential Medicines,however, the existing WHO listed dexamethasone products are not suitablefor BL treatment, as the higher dose of the present invention wouldrequire compounding vials that could lead to contamination and seriousor fatal infections in patients as well as to excipients such as benzylalcohol or parabens that reach toxic levels with compounding.

In yet further embodiments, the lymphocyte mediated disease is graftversus host disease (GvHD). GvHD is a medical complication following thereceipt of transplanted tissue from a genetically different person. GvHDcan occur even with autologous transplant, most likely caused by theprocessing and storage of the autologous cells such that thetransplanted cells then recognize the body as foreign. In GvHD, thewhite blood cells of the donor's immune system which remain within thedonated tissue (the graft) recognize the recipient (the host) as foreign(non-self). The white blood cells present within the transplanted tissuethen attack the recipient's body's cells, which leads to this condition.GvHD is commonly associated with stem cell transplants such as thosethat occur with bone marrow transplants. GvHD also applies to otherforms of transplanted tissues such as solid organ transplants. Thelymphodepletive action of the invention also contributes to the efficacyof these embodiments.

In other embodiments, the lymphocyte mediated disease is an allergicdisorder. This includes chronic and acute allergies. For instance, thepharmaceutical composition of the invention could be used in thetreatment of asthma. The lymphodepletive action of the invention alsocontributes to the efficacy of these embodiments.

In some embodiments, the pharmaceutical composition comprises apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition comprises a preservative and/or a chelatingagent. In some embodiments, the pharmaceutical composition comprises apreservative. Preferably, the preservative is a sulfite. In someembodiments, the pharmaceutical composition comprises a chelating agent,which may be EDTA.

In preferred embodiments, the glucocorticoid of the pharmaceuticalcomposition comprises dexamethasone. This may be in the form ofdexamethasone base, dexamethasone sodium phosphate or dexamethasoneacetate. Most preferably, the glucocorticoid is dexamethasone sodiumphosphate.

As noted above, and as defined by the claims, the pharmaceuticalcomposition of the invention is for use in the treatment of a lymphocytemediated disease. The treatment may comprise administering the dose ofthe pharmaceutical composition as a single acute dose.

Alternatively, the treatment comprises administering the dose of thepharmaceutical composition as a total dose given over about a 72 hourperiod.

The treatment of lymphocyte mediated diseases include administration ofthe compositions to patients in need of anti-inflammatory,immunosuppressive, lymphoablation, germinal center elimination, IL-2IL-7 IL-12 and/or IL-15 elevation, mesenchymal stem cell elevation,G-CSF increase, or neutrophil increase. Moreover, the treatment oflymphocyte mediated disease may result in detectable changes in PD-1 orPD-L1 or CTLA-4 expression.

As noted above, and as defined by the claims, the pharmaceuticalcomposition of the invention is for use in the treatment of a lymphocytemediated disease, wherein the treatment comprises administering a doseof the pharmaceutical composition to the patient. The pharmaceuticalcomposition may be administered intravenously (IV) or orally. Whenintravenous administration is performed, preferably, the dose isadministered as a single IV infusion over 0.25-2 hours. The infusedcomposition may be in normal or half-normal saline or Lactated Ringer'sor 5% Dextrose or another standard IV fluid solution. For oraladministration, the composition may be given as a single oral dose mixedwith a small amount of juice or sweetener.

In preferred embodiments, the pharmaceutical composition is provided asan aqueous glucocorticoid solution. The skilled person will appreciatethat this means that water is used as a solvent in the pharmaceuticalcompositions of these embodiments.

The pharmaceutical composition for the use according to the invention isadministered to deliver the glucocorticoid at a dose equivalent to atleast about 15 mg/kg, or at least about 16 mg/kg, or at least about 17mg/kg, or at least about 18 mg/kg, or at least about 19 mg/kg, or atleast about 20 mg/kg, or at least about 21 mg/kg, or at least about 22mg/kg, or at least about 23 mg/kg, or at least about 24 mg/kg, or atleast about 25 mg/kg, or at least about 26 mg/kg of a human equivalentdose (HED) of dexamethasone base. The dose of the pharmaceuticalcomposition can be defined as delivering the glucocorticoid at a doseequivalent to a value taken from a range of doses equivalent to HED ofdexamethasone base, where the range is defined by endpoints selectedfrom the above list of values, e.g. about 15 mg/kg-26 mg/kg, or about 18mg/kg-25 mg/kg (or any two values from the above list). In preferredembodiments, the subject is human, the glucocorticoid containsdexamethasone base, and the pharmaceutical composition is administeredto the human subject at a dose of between about 15.0 and about 21.0mg/kg of dexamethasone base.

The skilled person will understand that conventional methodology can beemployed to measure the lymphodepletion achieved by the invention. Forinstance, CD4+, CD8+, Tregs and/or B cells populations can be measuredafter the pharmaceutical composition has been administered, for instance48 hours after its administration. Flow cytometry is one exemplarymethod that may be used to perform the cell counts.

The skilled person will understand that this invention can be used inconjunction with other therapeutic approaches as described herein, forinstance chemotherapy and/or cell based therapies. In these embodiments,the subject may be administered chemotherapy. In these embodiments, thesubject may be administered a cell based therapy. However, mostembodiments of the invention do not involve chemotherapy or cell basedtherapies. Thus, in some embodiments, the subject is not administeredchemotherapy. In some embodiments, the subject is not administered cellbased therapies.

The mechanism of action of the invention is discussed in detail hereinand these mechanisms can form part of the distinctive features of theinvention in some instances, particularly where the mechanism opens up anew clinical situation (e.g. by allowing patient subgroups to beselected as the subjects).

SUMMARY OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Acute high dose dexamethasone eliminates binding niches in themouse spleen and secondary lymphatics. Shown are black and white scalebright field (top) and immunofluorescent (bottom) images of fresh thickspleen sections stained with FITC-PNA to quantitate germinal centersfrom mice administered IP human equivalent dose (HED) 9.3 mg kgdexamethasone base 96 hours before spleen harvest. The graph showscolumn plots of average germinal cell count per spleen area plusstandard area of the mean (SEM) for mice administered IP placebo controland IP HED 9.3 mg kg dexamethasone base 96 hours before spleen harvest.Control mice have significant FITC-PNA immunofluorescence, while micewho were injected with dexamethasone have almost no immunofluorescentsignal.

FIG. 2. Acute high dose dexamethasone dose-dependently eliminatesbinding niches in the mouse spleen. A graph of column plots of averageGerminal Center staining intensity measured using immunofluorescentstaining of fresh thick spleen sections stained with FITC-PNA is shown.Immunofluorescent intensity was calculated using thresholding andMetaMorph Image Analysis. Columns are average plus SEM. The mice wereadministered placebo, 3 mg/kg HED, 6 mg/kg HED, 9 mg/kg HED, or 12 mg/kgHED dexamethasone base 48 hours before spleen harvest. Germinal centerreduction is apparent at HED 6 mg/kg and is significantly reduced at HEDof 9 and 12 mg/kg doses.

FIG. 3. Acute high dose dexamethasone eliminates binding niches in therat spleen (MZ: marginal zone). Column plots of marginal zone widthsmeasured on 5 micron spleen sections from rats treated IV or PO withplacebo, 20 mg/kg (HED 3.23 mg/kg), 40 mg/kg (HED 6.45 mg/kg) or 80mg/kg (HED 12.9 mg/kg) dexamethasone base 48 hours before spleen harvestare shown. Marginal zone area was reduced at all dexamethasone doses,and was maximally inhibited at 12.9 mg/kg HED. n=5 per group. * p<0.05ANOVA (Dunnett's post-hoc) vs. Vehicle IV; † p<0.05 ANOVA (Dunnett'spost-hoc) vs. Vehicle PO; p<0.05 Student's t-test vs. Vehicle IV.

FIG. 4. Acute high dose dexamethasone eliminates binding niches in therat spleen. Column plots of the area per spleen of BCL-6 staining of 5micron fixed spleen sections as a measure of germinal center numbersgiven as average per section are shown. Rats were treated IV or PO withplacebo, 20 mg/kg (HED 3.23 mg/kg), 40 mg/kg (HED 6.45) or 80 mg/kg (HED12.9 mg/kg) dexamethasone base 48 hours before spleen harvest. Germinalcenter area was reduced at all dexamethasone doses, and was maximallyinhibited at 12.9 mg/kg HED. Groups 1-4 IV: 1=20 mg/kg (HED 3.23 mg/kg),2=40 mg/kg (HED 6.45 mg/kg), 3=80 mg/kg (HED 12.9 mg/kg), 4=Placebo.Groups 5-9 PO: 5=20 mg/kg (HED 3.23 mg/kg), 6=40 mg/kg (HED 6.45 mg/kg),7=80 mg/kg (HED 12.9 mg/kg), 8=Placebo.

FIG. 5. Acute high dose dexamethasone reduces thymic mass. Photographsshow size of thymus from placebo-treated murine subjects (topphotograph) and of thymus of murine subjects treated with a 6 mg/kg HEDdose of the pharmaceutical composition of the invention (lowerphotograph). The lower panel shows the thymus weight to body weightpercentage of the thymus of placebo-treated subjects (control) and ofsubjects treated with the pharmaceutical composition of the invention at3 mg/kg HED, 6 mg/kg HED, 9 mg/kg HED and 12 mg/kg HED.

FIG. 6. Acute high dose dexamethasone reduces rat lymphocyte number.Graphs of individual absolute lymphocyte numbers and averages measuredby complete blood count 48 hours after rats were treated IV (right) orPO (left) with placebo, 20 mg/kg (HED 3.23 mg/kg), 40 mg/kg (HED 6.45)or 80 mg/kg (HED 12.9 mg/kg) dexamethasone base are shown. Dexamethasonewas administered 48 hours before blood withdrawal. Significantlymphodepletion was observed at all doses vs. controls in rats whetherIV (right) or oral dosing (left). Doses are shown as HED (humanequivalent dose).

FIG. 7. Acute high dose dexamethasone does not reduce rat neutrophilnumber. Graphs of individual absolute neutrophil numbers and averagesmeasured by complete blood count 48 hours after rats were treated IV(right) or PO (left) with placebo, 20 mg/kg (HED 3.23 mg/kg), 40 mg/kg(HED 6.45) or 80 mg/kg (HED 12.9 mg/kg) dexamethasone base are shown.Data in FIGS. 3, 4, and 6 are from the same rats. Acute high dosedexamethasone has a lymphodepletion profile that is neutrophil sparing.Oral (left) and IV (right) doses were administered 1×48 hours beforeblood withdrawal. Doses are shown as HED (human equivalent dose).

FIG. 8. CD3 and CD4 positive lymphocytes. Graphs of individualCD3+(left) and CD4+(right) lymphocytes and averages measured by flowcytometry as relative counts and normalized to relative absolute countsusing complete blood counts 48 hours after mice were treated PO withplacebo, HED 3 mg/kg, HED 6 mg/kg, HED 9 mg/kg or HED 12.mg/kgdexamethasone base. Relative counts/ul=flow cytometry and complete bloodcount combined. Compared to control, in the 12 mg/kg group: 65%reduction in CD3+ cells; 75% reduction in CD4+ cells. Doses are shown asHED (human equivalent dose). A one-way ANOVA followed by Tukey's testwas incorporated to determine statistical significance between thetreatment groups; * p<0.05, ** p<0.01, *** p<0.001.

FIG. 9. Acute high dose dexamethasone reduces mouse CD8 positivelymphocytes and Tregs. Graphs of individual CD8+(left) and Treg (right)lymphocytes and averages measured by flow cytometry as relative countsand normalized to relative absolute counts using complete blood counts48 hours after mice were treated PO with placebo, HED 3 mg/kg, HED 6mg/kg, HED 9 mg/kg or HED 12.mg/kg dexamethasone base are shown. Treglymphocytes were identified by being CD3+CD4+CD25+FoxP3+. Relativecounts/ul=flow cytometry and complete blood counts combined. Compared tocontrol, in the 12 mg/kg group: 56% reduction in CD8+ cells; 78%reduction in mouse Tregs. Doses are shown as HED (human equivalentdose). A one-way ANOVA followed by post-hoc Tukey's test wasincorporated to determine statistical significance between the treatmentgroups; * p<0.05, ** p<0.01.

FIG. 10. Acute high dose dexamethasone reduces mouse NK cells and Blymphocytes. Graphs of individual natural killer (NK) cells (left) and Blymphocytes (right) and averages measured by flow cytometry as relativecounts and normalized to relative absolute counts using complete bloodcounts 48 hours after mice were treated PO with placebo, HED 3 mg/kg,HED 6 mg/kg, HED 9 mg/kg or HED 12.mg/kg dexamethasone base are shown.NK cells were identified by being CD3−CD49b+. B lymphocytes wereidentified by being CD3-B220+. Relative counts/ul=flow cytometry andcomplete blood counts combined. Compared to control, in the 12 mg/kggroup: 87% reduction in NK cells; 83% reduction B cells. Doses are shownas HED (human equivalent dose). A one-way ANOVA followed by post-hocTukey's test was incorporated to determine statistical significancebetween the treatment groups; * p<0.05, ** p<0.01; *** p<0.001.

FIG. 11. Acute high dose dexamethasone reduces mouse absolute lymphocytenumbers while sparing neutrophils. Graphs of individual absoluteneutrophils (left) and total lymphocytes (right) and averages measuredby complete blood counts 24-48 hours after mice were treated PO withplacebo, HED 3 mg/kg, HED 6 mg/kg, HED 9 mg/kg, HED 12.mg/kg, or HED17.5 mg/kg dexamethasone base are shown. Cells/ul=absolute numbersobtained from complete blood counts (CBC). Acute high dose dexamethasonecauses almost complete lymphoabalation at HED doses greater than 12mg/kg, but does not affect neutrophils. Acute high dose dexamethasonetherefore eliminates the need for transfusion, and provides a safer,non-toxic alternative to chemotherapeutic regimens. Doses are shown asHED (human equivalent dose).

FIG. 12. Acute high dose dexamethasone spares mouse RBCs and Platelets.Graphs of individual absolute RBC (left) and platelet (right) andaverages measured by complete blood counts 48 hours after mice weretreated PO with placebo, HED 3 mg/kg, HED 6 mg/kg, HED 9 mg/kg, HED12.mg/kg, or 17.5 mg/kg dexamethasone base are shown. Cells/ul=absolutenumbers obtained from CBC. Acute high dose dexamethasone does not affectRBCs or platelets, eliminates the need for transfusion, and thereforeprovides a safer, non-toxic alternative to chemotherapeutic regimens.Doses are shown as HED (human equivalent dose).

FIG. 13. Number of live hematopoietic stem cells measured 48 hours aftertreatment of naïve mice with placebo (vehicle), or low or high doses ofacute high dose dexamethasone are shown. Even high doses of acute highdose dexamethasone did not significantly alter the number of livehematopoietic stem cells. The non-myeloablative regimen represented byacute high dose dexamethasone could, therefore, eliminate the need fortransfusions of stem cells for hematopoietic recovery afterimmune-reset.

FIG. 14. Fifty percent (2 of 4) of human patients treated with 3 mg/kgdexamethasone base depleted CD3, CD4 and CD8 positive lymphocytes.Individual pre- and post-treatment, 48 hours after oral administrationof 3 mg/kg dexamethasone base to four human patients, values and lineplots of CD3+, CD4+, and CD8+ lymphocytes measured by flow cytometry areshown. Each patient's pre-treatment values are connected topost-treatment values by a connecting line. CD4+ cells are also CD3+.CD8+ cells are also CD3+.

FIG. 15. Twenty-five percent (1 of 4) of human patients treated with 3mg/kg dexamethasone base depleted Tregs and B lymphocytes. Line areindividual pre- and post-, 48 hours after oral administration of 3 mg/kgdexamethasone base to four human patients, values and line plots of Tregand B lymphocytes measured by flow cytometry. Each patient'spre-treatment values are connected to post-treatment values by aconnecting line. Tregs are identified by being CD3+CD4+CD25+FoxP3+. Blymphocytes are identified by being CD3−CD19+.

FIG. 16. Seventy-five percent (3 of 4) of human patients treated with 3mg/kg dexamethasone base depleted NK cells while hematopoietic stemcells were spared. Line are individual pre- and post-treatment, 48 hoursafter oral administration of 3 mg/kg dexamethasone base to four humanpatients, values and line plots of NK cells and Hematopoietic Stem Cells(HSCs) measured by flow cytometry. Each patient's pre-treatment valuesare connected to post-treatment values by a connecting line. NK cellsare identified by being CD3−CD16/56+. HSCs are identified by beingCD34+CD38−.

FIG. 17. 100% of human patients treated with 3 mg/kg dexamethasone baseshowed increased serum IL-2 and/or IL-15 levels, but no elevation inIL-6. Column plots of each patients pre- and post-treatment, 48 hoursafter oral administration of 3 mg/kg dexamethasone base to four humanpatients, plasma levels of interleukin 2 and interleukin 15 measured byProCartaPlex-9 plx Luminex assay. FIG. 14, FIG. 15, FIG. 16 and FIG. 17show data from the same four human patients.

FIG. 18. Oral administration of 3 mg/kg dexamethasone base increasedbone marrow MSC number 48 hours later. Column plots of data from 31historical naïve control humans plus standard deviation, and two humanpatients treated with 3 mg/kg dexamethasone base 48 hours beforeaspiration of concentrated bone marrow from the ileac crest using aMarrowCellution™ needle. Plots show bone marrow CFU/ml+/− stdev. Bonemarrow was added directly to colony forming unit assay fibroblast(CFU-F) media without further manipulation 24 hours after harvest andshipment at controlled room temperature. CFU-F colony number is ameasure of mesenchymal stem cell (MSC) number in the starting material.48 hours after oral administration of 3 mg/kg dexamethasone base, ileaccrest bone marrow MSC numbers appear about twice as high as 31historical controls. 3 mg/kg oral dexamethasone base increases humanbone marrow CFU-F per ml 48 hours later compared to 31 historicalcontrols aspirated using the same MarrowCellution™ needle as forpatients M and P.

FIG. 19. Comparison of a 12 mg/kg and 17-18 mg/kg dexamethasone baseoral dose on day −2 to a single dose of Cyclophosphamide 166 mg/kg (HED500 mg/m2) and Fludarabine 10 mg/kg on day −5 combined with 12 mg/kg or17-18 mg/kg dexamethasone base on day −2, and to 2 days of repeatCyclophosphamide 166 mg/kg on day −5 and −4 and 4 days of Fludarabine 10mg/kg (HED 30 mg/m2) on days −5, −4, −3, −2. Shown is a graph ofindividual absolute lymphocytes and averages (left) measured by completeblood counts 48 hours after mice were treated IP with PBS (Vehicle), orwith repeat IP Cyclophosphamide 166 mg/kg on day −5 and −4 and 4 days ofIP Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3, −2 (Flu+Cy),or with a single IP dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2)and IP Fludarabine 10 mg/kg both on day −5 and then with oral 12 mg/kgor 17-18 mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12 mg/kg);Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)). Also shown(right) is a representation of the dosing schedules in mice of theseregimens.

FIG. 20. A single dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2) andFludarabine 10 mg/kg on day −5 combined with 12 mg/kg or 17-18 mg/kgdexamethasone base on day −2 equivalently lymphodepleted CD3+ and CD4+lymphocytes compared to 2 days of repeat Cyclophosphamide 166 mg/kg onday −5 and −4 and 4 days of Fludarabine 10 mg/kg (HED 30 mg/m2) on days−5, −4, −3, −2. Shown are graphs of individual CD3+(left) andCD4+(right) lymphocytes and averages measured by flow cytometry asrelative counts and normalized to relative absolute counts usingcomplete blood counts 48 hours after mice were treated IP with PBS(Vehicle), or with repeat IP Cyclophosphamide 166 mg/kg on day −5 and −4and 4 days of IP Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3,−2 (Flu+Cy), or with a single IP dose of Cyclophosphamide 166 mg/kg (HED500 mg/m2) and IP Fludarabine 10 mg/kg both on day −5 and then with oral12 mg/kg or 17-18 mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12mg/kg); Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)). On both theCD3+ plot (left) and the CD4+ plot (right), the 12 mg/kg or 17-18 mg/kgdexamethasone base data are shown in the right-hand columns of each(relative counts are ‘92’, ‘71’, ‘37’ and ‘25’).

FIG. 21. A single dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2) andFludarabine 10 mg/kg on day −5 combined with 12 mg/kg or 17-18 mg/kgdexamethasone base on day −2 equivalently lymphodepleted CD8+lymphocytes and Tregs compared to 2 days of repeat Cyclophosphamide 166mg/kg on day −5 and −4 and 4 days of Fludarabine 10 mg/kg (HED 30 mg/m2)on days −5, −4, −3, −2. Shown are graphs of individual Treg (right) andCD8+ lymphocytes (left) and averages measured by flow cytometry asrelative counts and normalized to relative absolute counts usingcomplete blood counts 48 hours after mice were treated IP with PBS(Vehicle), or with repeat IP Cyclophosphamide 166 mg/kg on day −5 and −4and 4 days of IP Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3,−2 (Flu+Cy), or with a single IP dose of Cyclophosphamide 166 mg/kg (HED500 mg/m2) and IP Fludarabine 10 mg/kg both on day −5 and then with oral12 mg/kg or 17-18 mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12mg/kg); Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)). On both theCD8+ plot (left) and the CD4+ plot (right), the 12 mg/kg or 17-18 mg/kgdexamethasone base data are shown in the right-hand columns of each(relative counts are ‘33’, ‘1.4’, ‘0.2’ and ‘0.5’).

FIG. 22. A single dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2) andFludarabine 10 mg/kg on day −5 combined with 12 mg/kg or 17-18 mg/kgdexamethasone base on day −2 equivalently lymphodepleted NK cells and Blymphocytes compared to 2 days of repeat Cyclophosphamide 166 mg/kg onday −5 and −4 and 4 days of Fludarabine 10 mg/kg (HED 30 mg/m2) on days−5, −4, −3, −2. Shown are graphs of individual B lymphocytes (left) andNK cell lymphocytes (right) and averages measured by flow cytometry asrelative counts and normalized to relative absolute counts usingcomplete blood counts 48 hours after mice were treated IP with PBS(Vehicle), or with repeat IP Cyclophosphamide 166 mg/kg on day −5 and −4and 4 days of IP Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3,−2 (Flu+Cy), or with a single IP dose of Cyclophosphamide 166 mg/kg (HED500 mg/m2) and IP Fludarabine 10 mg/kg both on day −5 and then with oral12 mg/kg or 17-18 mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12mg/kg); Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)). On both theB cell plot (left) and the NK cell plot (right), the 12 mg/kg or 17-18mg/kg dexamethasone base data are shown in the right-hand columns ofeach (relative counts are ‘111’ and ‘58’ for B cells; not shown for NKcells).

FIG. 23. A single dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2) andFludarabine 10 mg/kg on day −5 combined with 12 mg/kg or 17-18 mg/kgdexamethasone base on day −2 equivalently lymphodepleted absolutelymphocytes, but spared neutrophils, compared to 2 days of repeatCyclophosphamide 166 mg/kg on day −5 and −4 and 4 days of Fludarabine 10mg/kg (HED 30 mg/m2) on days −5, −4, −3, −2. Shown are graphs ofindividual absolute neutrophils (left) and absolute lymphocytes (right)and averages measured by complete blood counts 48 hours after mice weretreated IP with PBS (Vehicle), or with repeat IP Cyclophosphamide 166mg/kg on day −5 and −4 and 4 days of IP Fludarabine 10 mg/kg (HED 30mg/m2) on days −5, −4, −3, −2 (Flu+Cy), or with a single IP dose ofCyclophosphamide 166 mg/kg (HED 500 mg/m2) and IP Fludarabine 10 mg/kgboth on day −5 and then with oral 12 mg/kg or 17-18 mg/kg dexamethasonebase on day −2 (Flu+Cy+AVM0703 (12 mg/kg); Flu+Cy+AVM0703 (17 mg/kg)),or with oral 12 mg/kg or 17-18 mg/kg dexamethasone base (AVM0703 (12mg/kg); AVM0703 (17 mg/kg)). On both the neutrophil plot (left) and thelymphocyte plot (right), the 12 mg/kg or 17-18 mg/kg dexamethasone basedata are shown in the right-hand columns of each (relative counts are‘321’, ‘605’, ‘521’ and ‘88’).

FIG. 24. A single dose of Cyclophosphamide 166 mg/kg (500 mg/m2) andFludarabine 10 mg/kg (HED 30 mg/m2) on day −5 combined with 12 mg/kg or17-18 mg/kg dexamethasone base on day −2 spared red blood cells (RBCs)and platelets. Shown are graphs of individual absolute platelet andabsolute RBCs and averages measured by complete blood counts 48 hoursafter mice were treated IP with PBS (Vehicle), or with repeat IPCyclophosphamide 166 mg/kg on day −5 and −4 and 4 days of IP Fludarabine10 mg/kg (HED 30 mg/m2) on days −5, −4, −3, −2 (Flu+Cy), or with asingle IP dose of Cyclophosphamide 166 mg/kg (HED 500 mg/m2) and IPFludarabine 10 mg/kg both on day −5 and then with oral 12 mg/kg or 17-18mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12 mg/kg);Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)). On both theRBC plot (left) and the platelet plot (right), the 12 mg/kg or 17-18mg/kg dexamethasone base data are shown in the right-hand columns ofeach (relative counts are ‘10’, ‘10’, ‘348’ and ‘373’).

FIG. 25. A single dose of Cyclophosphamide 166 mg/kg (500 mg/m2) andFludarabine 10 mg/kg on day −5 combined with 12 mg/kg or 17-18 mg/kgdexamethasone base on day −2 spared body weight, a measure of toxicity,compared to 2 days of repeat Cyclophosphamide 166 mg/kg on day −5 and −4and 4 days of Fludarabine 10 mg/kg on days −5, −4, −3, −2. Shown (left)are graphs of individual body weight differences and averages calculatedby subtracting body weight 48 hours after mice were treated IP with PBS(Vehicle), or with repeat IP Cyclophosphamide 166 mg/kg on day −5 and −4and 4 days of IP Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3,−2 (Flu+Cy), or with a single IP dose of Cyclophosphamide 166 mg/kg (HED500 mg/m2) and IP Fludarabine 10 mg/kg both on day −5 and then with oral12 mg/kg or 17-18 mg/kg dexamethasone base on day −2 (Flu+Cy+AVM0703 (12mg/kg); Flu+Cy+AVM0703 (17 mg/kg)), or with oral 12 mg/kg or 17-18 mg/kgdexamethasone base (AVM0703 (12 mg/kg); AVM0703 (17 mg/kg)) frompretreatment body weights. The acute high dose dexamethasone group isnot associated with body weight loss unlike the chemotherapy groups.Acute high dose dexamethasone therefore provides a similarlymphodepletion effect as chemotherapy but with no associated toxicity.Also shown (right) is a representation of the dosing schedules in theseregimens.

DETAILED DESCRIPTION

Cytotoxic chemotherapeutic agents trigger cell death via mechanisms ormeans that are not receptor mediated. Cytotoxic chemotherapeutic agentstrigger cell death by interfering with functions that are necessary forcell division, metabolism, or cell survival. Because of this mechanismof action, cells that are growing rapidly (which means proliferating ordividing) or are active metabolically will be killed preferentially overcells that are not. The status of the different cells in the body asdividing or as using energy (which is metabolic activity to supportfunction of the cell) determines the dose of the chemotherapeutic agentthat triggers cell death. The skilled person will appreciate that theglucocorticoid that is utilized in this invention is not a cytotoxicchemotherapeutic. Cytotoxic chemotherapeutic agents non-exclusivelyrelates to alkylating agents, anti-metabolites, plant alkaloids,topoisomerase inhibitors, antineoplastics and arsenic trioxide,carmustine, fludarabine, IDA ara-C, myalotang, GO, mustargen,cyclophosphamide, gemcitabine, bendamustine, total body irradiation,cytarabine, etoposide, melphalan, pentostatin and radiation.

The present invention pertains to pharmaceutical compositions comprisinga glucocorticoid for use in the treatment of diseases by immunoablation.In particular, the compositions of the invention may be for use in thetreatment of diseases that are mediated by immune cells such aslymphocytes. The treatment comprises administering a dose of thepharmaceutical composition to the patient to deliver the glucocorticoidat a dose equivalent to about 15 to about 26 mg/kg human equivalent dose(HED) of dexamethasone base.

As used herein, the term glucocorticoid includes glucocorticoid receptoragonists and any compound that binds to the glucocorticoid receptor.Such compounds relate to, but are not limited to, dexamethasone,dexamethasone containing agents, hydrocortisone, methyl predisone,prednisone, corticone, budesonide, betamethasone and beclomethasone.Other glucocorticoids include prednisolone, mometasone furoate,Triamcinolone Acetonide and methylprednisolone. Glucocorticoids furtherinclude glucocorticoid receptor modulating agonists. Additionally,selective glucocorticoid receptor agonists may be used in thepharmaceutical compositions disclosed herein. Such agonists ormodulators include for example, selective glucocorticoid receptormodulators (SEGRMs) and selective glucocorticoid receptor agonists(SEGRAs). Glucocorticoids, glucocorticoid receptor modulators andselective glucocorticoid receptor agonists (SEGRAs) that may be utilizedin the herein disclosed methods and compositions are well know to thoseskilled in the art.

Glucocorticoids and glucocorticoid-receptor (GR) modulating agents exerttheir effects through both membrane glucocorticoid receptors andcytoplasmic GRs which activate or repress gene expression. Some of thedesirable lymphodepletion effects of the glucocorticoids and GRmodulating agents appear to be mediated via membrane GRs or othernon-genomic effects in addition to their genomic effects. Interestingly,co-treatment with dexamethasone has been shown to be able to reduceglucocorticoid resistance (Serafin et al., 2017).

The effects of glucocorticoids are complex and depend on each specificglucocorticoid's affinity for the GR and mineralocorticoid receptor(MR). Additionally, there are now 9 known isoforms of the cytosolic GRand additional membrane expressed GR receptors that have been identifiedbut which are not fully characterized. Glucocorticoids have beenreported to have varied effects on lymphocyte levels, depending on theconcentration of the glucocorticoid administered and the duration oftreatment. In general, at low doses typically used for chronic therapy,glucocorticoids have been reported to redistribute lymphocytes from theperipheral blood into the bone marrow, at medium doses glucocorticoidshave been reported to cause leukocytosis thought to be a redistributionof leukocytes from the bone marrow, spleen and thymus into theperipheral blood, and at high doses glucocorticoids have a lymphotoxicaction on lymphocytes by triggering apoptosis and necroptosis. Theduration of effect also depends on the dose level, for instance Fauci etal (1976) reports a single oral 0.24 mg/kg dexamethasone dose suppressesperipheral blood T and B lymphocytes 80% with recovery beginning at 12hours and normal levels by 24 hours. However, the present inventiondemonstrates that acute oral doses of 3 mg/kg or greater are necessaryto reduce peripheral blood T and B cells 24-48 hours afteradministration, with return to baseline levels occurring around 5 to 14days after dosing.

The desired in vivo effects of exemplary glucocorticoids would includereductions in germinal center and marginal zones in secondarylymphatics, direct tumor killing of some cancers particularly; multiplemyeloma, renal cell carcinoma, leukemia and lymphoma, non-small celllung cancer (NSCLC), prostate and breast cancer; depletion of allperipheral blood lymphocyte types, lack of lymphocyte redistribution tothe BM or other organs, and elevation of plasma cytokines includingIL-2, and/or IL-7, and/or IL-12, and/or IL-15 to levels preferably of 20pg/ml or greater, among others. Exemplary glucocorticoids do not elevateplasma levels of IL-6, one of the major contributors to ACT inducedcytokine release syndrome (CRS). Exemplary glucocorticoids do notelevate plasma levels of GM-CSF, one of the major contributors to ACTinduced neuroedema. Acute doses of dexamethasone of about HED 6 mg/kgand above reduce germinal centers and marginal zones in secondarylymphatics; acute doses of dexamethasone of about 1.6 mg/kg HED in a 48hour period have about 50% direct tumor killing against multiple myelomaand other cancer cell lines which is maintained but not increased withdoses up to about 12 mg/kg HED; acute doses of dexamethasone of greaterthan about HED 3 mg/kg are required for lymphodepletion demonstrated bythe observation that 50% of patients treated with 3 mg/kg HED showedlymphocytosis (FIG. 14); plasma IL-2 and IL-15 cytokine elevations areobserved at doses of dexamethasone base of about HED 3 mg/kg or higher(FIG. 17). Based on the desired in vivo effects in the indicationsdisclosed in this application, the most preferred acute dexamethasonebase doses, which can be converted to equivalent doses of otherglucocorticoids based on known calculators or as disclosed in thisdescription, will be most likely about HED 9 mg/kg and above.

A single high dose of glucocorticoid can be given as an oraladministration or about a one hour IV infusion. A total dose may begiven as repetitive IV or oral doses in any quantity such that the totaldose, e.g. of dexamethasone, is about 15 mg/kg to about 26 mg/kg withinabout a 24 to about a 72 hour period.

Equivalent doses of another glucocorticoid or glucocorticoid receptormodulating agent can be readily and easily calculated using publiclyavailable corticoid conversion algorithms, preferablyhttp://www.medcalc.com. For instance, 3 to 12 mg/kg dexamethasoneconverts to 19 to 75 mg/kg prednisone. Since prednisone's biologichalf-life is about 20 hours, while dexamethasone's biologic half-life isabout 36 to 54 hours. Therefore, prednisone would be dosed between 19 to75 mg/kg every 24 hours for equivalent biologic dosing. Morespecifically, a 12 mg/kg dose of dexamethasone corresponds to 1) a 75mg/kg dose of prednisolone that would require repeat dosing of about twoto about three doses every 24 hours. A 10 mg/kg dose of betamethasone isabout 12 mg/kg dexamethasone and has a pharmacodynamic (biologic)half-life similar to dexamethasone. However, betamethasone reduces RBCat doses of about 24 mg/50 kg (Gaur 2017).

DEX (dexamethasone base) doses in the examples in the presentapplication are given as human equivalent doses (HED). AVM0703 (alsoreferred to as AugmenStem™ or PlenaStem™) in the examples given is Dex(dexamethasone base) as dexamethasone sodium phosphate in a proprietarybuffer.

Methods for calculating the human equivalent dose (HED) are known in theart. For example the FDA's Centre for Drug Evaluation and Research(CDER) issued a highly-cited guidance document in 2005 (U.S Departmentof Health CDER, 2005), which sets out the established algorithm forconverting animal doses to HED based on body surface area (the generallyaccepted method for extrapolating doses between species) at Table 1 onpage 7 of that document. For reference, Table 1 is reproduced below. Theskilled person understands that the animal dose in mg/kg, explainedbelow, the HED is calculated easily using the standard conversionfactors in the right hand columns of Table 1:

TABLE 1 Conversion of Animal Doses to Human Equivalent Doses Based onBody Surface Area To Convert To Convert Animal Dose Animal Dose in inmg/kg to HED^(a) in mg/kg to Dose mg/kg, Either: in mg/m², Divide AnimalMultiply Animal Species Multiply by k_(m) Dose By Dose By Human 37 — —Child (20 kg)^(b) 25 — — Mouse 3 12.3 0.08 Hamster 5 7.4 0.13 Rat 6 6.20.16 Ferret 7 5.3 0.19 Guinea pig 8 4.6 0.22 Rabbit 12 3.1 0.32 Dog 201.8 0.54 Primates: Monkeys^(c) 12 3.1 0.32 Marmoset 6 6.2 0.16 Squirrelmonkey 7 5.3 0.19 Baboon 20 1.8 0.54 Micro-pig 27 1.4 0.73 Mini-pig 351.1 0.95 ^(a)Assumes 60 kg human. For species not listed or for weightsoutside the standard ranges, HED can be calculated from the followingformula: HED = animal dose in mg/kg × (animal weight in kg/human weightin kg)^(0.33). ^(b)This k_(m) value is provided for reference only sincehealthy children will rarely be volunteers for phase 1 trials. ^(c)Forexample, cynomolgus, rhesus, and stumptail.

Doses described herein can be presented as a “weight based dose” or as a“body surface area (BSA) based dose.” A weight based dose is a dose thatis administered to a patient that is calculated based on the weight ofthe patient, e.g., mg/kg. A BSA based dose is a dose that isadministered to a patient that is calculated based on the surface areaof the patient, e.g., mg/m2. The two forms of dose measurement can beconverted within the context of human dosing by multiplying the weightbased dose by 37 or dividing the BSA based dose by 37 as shown in Table1 above.

The terms “subject” and “patient” are used interchangeably herein, andrefer to a human or animal.

Dexamethasone, like the other glucocorticoid steroids at equivalentdoses, inhibits the formation and proliferation of germinal centers inthe lymph tissues and lymphodepletes peripheral blood. The doses ofglucocorticoid, particularly dexamethasone, preferably achieve greaterthan 75% lymphodepletion. More preferably, the doses of glucocorticoid,particularly dexamethasone, achieve greater than 80% lymphodepletion.Most preferably, the dose of glucocorticoid, particularly dexamethasone,achieves greater than 95% lymphodepletion. The skilled person willunderstand that lymphodepletion can be measured readily by measuringcomplete blood counts (CBCs).

Dexamethasone and other preferred glucocorticoids spare neutrophils anddo not inhibit neutrophil function (Schleimer R P, J Pharmacol Exp Ther1989; 250:598-605), and spare red blood cells (RBCs), platelets,mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC).Neutrophil sparing in humans is an absolute neutrophil count (ANC)greater than 500 per mm³. By sparing neutrophils, RBCs and platelets,lymphoablating glucocorticoids would reduce or eliminate the need fortransfusions. Lymphoablating glucocorticoids also spare bone marrowmesenchymal stem cells (MSCs) and do not affect the capacity of bonemarrow MSCs to differentiate towards chondrocytes, osteocytes oradipocytes. Lymphoablating glucocorticoids also increase the endogenousnumber of BM MSCs or their ex vivo survival in both humans and horses.Lymphoablating glucocorticoids increase plasma IL-2, IL-7, IL-12 andIL-15 levels, but not IL-6 or GM-CSF levels. In some embodiments of theinvention, the subject is selected before treatment and/or assessedafter treatment, based on measurements of the plasma levels of one ormore of these cytokines.

Dexamethasone is approved for use with an initial dosage ofdexamethasone sodium phosphate injection that varies from 0.5 to 9 mg aday depending on the disease being treated, which is a daily dose of0.01 to 0.18 mg/kg based on a 50 kg BW. In less severe diseases doseslower than 0.5 mg may suffice, while in severe diseases doses higherthan 9 mg may be required. There is a tendency in current medicalpractice to use high (pharmacologic) doses of corticosteroids for thetreatment of unresponsive shock. For cerebral edema Dexamethasone sodiumphosphate injection is generally administered initially in a dosage of10 mg intravenously followed by four mg every six hours intramuscularlyuntil the symptoms of cerebral edema subside. This total dose wouldcorrespond to a total 24 hour dose of about 0.34 to 0.48 mg/kg and atotal 72 hour dose of 0.8 to 1.12 mg/kg in 72 hours, which is not aneffective dose according to the present invention, which uses dosesbetween about 15 mg/kg and about 26 mg/kg.

For acute allergic disorders, dexamethasone sodium phosphate injection,USP 4 mg/mL; is recommended: first day, 1 or 2 mL (4 or 8 mg),intramuscularly, then Dexamethasone sodium phosphate tablets, 0.75 mg;second and third days, 4 tablets in two divided doses each day; fourthday, 2 tablets in two divided doses; fifth and sixth days, 1 tablet eachday; seventh day, no treatment; eighth day, follow-up visit.Dexamethasone has been used in the emergency room for severe acutepediatric asthma at 2 mg/kg, a dose which is below the glucocorticoiddoses as defined in this invention. Dexamethasone has also been used at6 mg/kg to treat pediatric acute lymphoblastic leukemia, a dose which isbelow the glucocorticoid doses as defined in this invention.

Conventional formulations of glucocorticoids such as dexamethasone maybe unsuitable for use in the therapeutic applications of the presentinvention. For instance, dexamethasone sodium phosphate (DSP) iscurrently available in low dose (2-4 mg/ml) and low volume formulations(e.g. APP Pharmaceuticals, Mylan), which contain antimicrobialpreservatives such as benzyl alcohol (BA) and propyl paraben (PP). Thetarget dose of DSP required for performing complete lymphoablation wouldentail the use of multiple vials resulting in overdoses of excipients.Exceeding WHO acceptable daily intake (ADI) of both benzyl alcohol andpropyl paraben has been associated with genotoxicity and increased riskof cancer (Darbre et al., 2014), reproductive toxicity (Aker et al.,2016), increased risks of allergic disease (Savage et al., 2012; Spanieret al., 2014), and neonatal CNS dysfunctions (Medicines Agency, 2017).Moreover, with commercially available DSP package inserts, seriousneuropsychiatric effects occur in about 6% of patients who receivesteroids (Malmegrim et al., 2017). As the present invention involves theadministration of high doses of glucocorticoids, formulations with lowlevels of potentially toxic preservatives, or formulations without toxicpreservatives, should be used. Preferably, the preservative is anantioxidant.

The pharmaceutical composition of the invention may include apreservative (e.g. an antioxidant) additive such as sodium sulfite tomaintain the stability of the composition. Sulfites are also widely usedas preservative and antioxidant additives in the pharmaceuticalindustries. Exposure to such sulfites has been reported to induce arange of adverse clinical effects in sensitive individuals, ranging fromdermatitis, urticaria, flushing, hypotension and abdominal pain tolife-threatening anaphylactic and asthmatic reactions. Sulfite-inducingsymptoms range from mild in some individuals, to severe in others, andin some individuals the reactions can be life threatening. In preferredembodiments, where sodium sulfite is included as an antioxidant, theconcentration is between 0-70 ppm Sodium Sulfite (Anhydrous).

Antioxidants may be added in amounts that are reduced from those levelstypically employed in glucocorticoid containing compositions therebyreducing the toxicity and adverse side effects associated with the useof such antioxidants. In some instances, the formulations of theinvention may lack the addition of antioxidants.

As used herein, antioxidants are those excipients that delay or inhibitthe oxidation process of molecules thereby increasing the stability ofthe composition. Antioxidants that may be used include, for example,ascorbic acid, acetylcysteine, butylhydroxyanisol, cysteinehydrochloride, dithionite sodium, gentisic acid, glutamate monosodium,glutathione, formaldehyde sulfoxylate sodium, methionine,monothioglycerol, propyl gallate, sulfites, sodium thioglycolate,α-thioglycerol, tocopherol alpha, alpha tocopherol hydrogen succinateand thioglycolate sodium.

In addition to an active glucocorticoid and antioxidant, additionalcomponents well known to those of skill in the art may be included inthe pharmaceutical compostions disclosed herein. Pharmaceuticalcompositions may be prepared using a pharmaceutically acceptable“carrier” composed of materials that are considered safe and effective.“Pharmaceutically acceptable” refers to molecular entities andcompositions that are “generally regarded as safe”, e.g., that arephysiologically tolerable and do not typically produce an allergic orsimilar untoward reaction, such as gastric upset and the like, whenadministered to a human. In some embodiments, this term refers tomolecular entities and compositions approved by a regulatory agency ofthe US federal or a state government, as the GRAS list under section204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that issubject to premarket review and approval by the FDA or similar lists,the U.S. Pharmacopeia or another generally recognized pharmacopeia foruse in animals, and more particularly in humans.

The term “carrier” refers to diluents, binders, lubricants anddisintegrants. Those with skill in the art are familiar with suchpharmaceutical carriers and methods of compounding pharmaceuticalcompositions using such carriers.

The pharmaceutical compositions provided herein may include one or moreexcipients, e.g., solvents, solubility enhancers, suspending agents,buffering agents, isotonicity agents, antioxidants or antimicrobialpreservatives. When used, the excipients of the compositions will notadversely affect the stability, bioavailability, safety, and/or efficacyof the active ingredients, ie., glucocorticoids, used in thecomposition. Thus, the skilled person will appreciate that compositionsare provided wherein there is no incompatibility between any of thecomponents of the dosage form. Excipients may be selected from the groupconsisting of buffering agents, solubilizing agents, tonicity agents,chelating agents, antioxidants, antimicrobial agents, and preservatives.

The pharmaceutical composition of the invention may include a chelatingagent which is used to sequester and decrease the reactivity of metalions that may be present in the compositions. Possible chelators arecalcium disodium EDTA 0.01-0.1% (EDTA=Ethylenediaminetetra acetic acidor Edetate), Disodium EDTA 0.01-0.11%, Sodium EDTA 0.20%, CalciumVersetamide Sodium 2.84%, Calteridol 0.023%, DTPA 0.04-1.2%(Diethylenetriaminepenta acetic acid). In a preferred embodiment theconcentration of Disodium EDTA (Edetate) is between 0 and 500 ppm.

As noted in International patent application PCT/US2018/025517 (filed 30Mar. 2018), glucocorticoids can also be used as a preconditioning agent,in conjunction with adoptive cell therapies (ACT). Glucocorticoids,particularly dexamethasone dosed between about 3 mg/kg and about 26mg/kg single acute dose about 12 to about 72 hours prior to cellimmunotherapy administration or total dose of about 15 mg/kg to about 26mg/kg given between about 12 to about 72 hours of cell therapyadministration increases plasma IL-2 and IL-15 levels.

Glucocorticoids, particularly dexamethasone dosed between about 15 mg/kgand about 26 mg/kg single acute dose or total dose of about 15 mg/kg toabout 26 mg/kg given over about a 72 hour period, either alone, or incombination with reduced intensity cytotoxic preconditioning can beuseful for the treatment of autoimmune diseases. For the treatment ofautoimmune disease an ACT could be targeted to the immune cells drivingthe disease in an effort to eradicate the autoimmune recognizing cells.Additionally, for autoimmune diseases the ACT could be a Treg targetedby a CAR or TCR or expressed antibody for an antigen expressedspecifically or selectively by the region or organ of the body where theautoimmune attack goes on. The Tregs could non-exclusively relate toCD4+Tregs, CD4+CD45RA+Tregs, CD4+CD25+CD45RA+Tregs, FoxP3+Tregs,CD4+CD25+FoxP3+CD152+Tregs, CD4+CD25+CD152+Tregs, CD8+Tregs,CD8+CD28−Tregs, CD4+CD25int/high, CD127low, CTLA4+, GITR+, FoxP3+,CD127low, CD4+CD25−⁻ induced Tregs, or Type I T regs.

“Natural” regulatory T cells originally recognised by their constitutiveexpression of CD4 and CD25 can be further defined by expression of thetranscription factor foxP3 and surface CD152. Their generation and someof their suppressive activity is dependent on TGF-beta, and it has beenshown that they can induce IDO in appropriate DCs by CD152 mediatedligation of CD80/86. Anergic CD4+ T cells generated by antigenstimulation in the absence of costimulation seem to be characterised byan intrinsic raising of their threshold for antigen stimulation that maybe maintained by expression of E3 ubiquitin ligases such as GRAIL, c-cbland Itch. Anergic cells can act as regulatory T cells by competing atthe sites of antigen presentation and adsorbing out stimulatorycytokines such as IL-2. Tr1 cells represent an induced subset of CD4helper T cells that are dependent on IL-10 for their differentiation andfor some of their regulatory properties. They do not express foxP3 butmay express markers associated with Th2 cells and repressor of GATA(ROG). Like natural Tregs, they express high levels of surface CD152 andcan induce IDO and trypophan catabolism in appropriate DCs. CD8+CD28−suppressor T (Ts) cells were first characterised in human, but haverecently also been demonstrated in rodents. Like Tr1 cells, they areinduced in the presence of IL-10, and IL-10 may be involved in thedownregulation of dendritic cell costimulation and the upregulation ofILT-3 and ILT-4 (in human DC) that seem to play an important role inpresenting antigen to tolerise further cohorts of T cells.

Regulatory T cells (Tregs) play an important role in maintaining immunehomeostasisl. Tregs suppress the function of other T cells to limit theimmune response. Alterations in the number and function of Tregs hasbeen implicated in several autoimmune diseases including multiplesclerosis, active rheumatoid arthritis, and type 1 diabetes. High levelsof Tregs have been found in many malignant disorders including lung,pancreas, and breast cancers. Tregs may also prevent antitumor immuneresponses, leading to increased mortality.

Two major classes of Tregs have been identified to date: CD4 and CD8Tregs. CD4 Tregs consist of two types, “natural” Tregs (nTregs) thatconstitutively express CD25 and FoxP3, and so-called adaptive orinducible Tregs (iTregs).

Natural Tregs (nTregs) originate from the thymus as CD4⁺ cellsexpressing high levels of CD25 together with the transcription factor(and lineage marker) FoxP3. nTregs represent approximately 5-10% of thetotal CD4⁺ T cell population, and can first be seen at thesingle-positive stage of T lymphocyte development. They are positivelyselected thymocytes with a relatively high avidity for self-antigens.(Fehérvari Z, Sakaguchi S. Development and function of CD25⁺CD4⁺regulatory T cells. Curr Opin Immunol. 2004; 16:203-208.)

The signal to develop into Treg cells is thought to come frominteractions between the T cell receptor and the complex of MHC II withself peptide expressed on the thymic stroma. nTregs are essentiallycytokine independent.

Adaptive or inducible Tregs originate from the thymus as single-positiveCD4 cells. They differentiate into CD25 and FoxP3 expressing Tregs(iTregs) following adequate antigenic stimulation in the presence ofcognate antigen and specialized immunoregulatory cytokines such asTGF-β, IL-10, and IL-4. (Chatenoud L, Bach J F. Adaptive humanregulatory T cells: myth or reality? J Clin Invest. 2006;116:2325-2327.)

FoxP3 is currently the most accepted marker for Tregs, although therehave been reports of small populations of FoxP3⁻ Tregs. The discovery oftranscription factor FoxP3 as a marker for Tregs has allowed scientiststo better define Treg populations leading to the discovery of additionalTreg markers including CD127.

Glucocorticoids, particularly dexamethasone dosed between about 15 mg/kgand about 26 mg/kg single acute dose or total dose of about 15 mg/kg toabout 26 mg/kg given over about a 72 hour period, either alone or incombination with reduced intensity cytotoxic chemotherapy or radiationcan be useful for the treatment of residual HIV disease, and for thetreatment of germinal center lymphomas such as Burkitt's Lymphoma.

Follicular helper CD4 T cells, T_(FH), residing in B-cell follicleswithin secondary lymphoid tissues, are readily infected by AIDS virusesand are a major source of persistent virus despite relative control ofviral replication. This persistence is due at least in part to arelative exclusion of effective antiviral CD8 T cells from B-cellfollicles. AIDS virus persistence in individuals under effective drugtherapy or those who spontaneously control viremia remains an obstacleto definitive treatment. Infected follicular helper CD4 T cells, T_(FH),present inside B-cell follicles represent a major source of thisresidual virus. While effective CD8 T-cell responses can control viralreplication in conjunction with drug therapy or in rare casesspontaneously, most antiviral CD8 T cells do not enter B-cell follicles,and those that do fail to robustly control viral replication in theT_(FH) population. Thus, these sites are a sanctuary and a reservoir forreplicating AIDS viruses. Lymphodepletion and reduction of germinalcenters and marginal zones in the spleen would force residual HIVinfected cells into the blood stream where they could be killed byexisting therapies. Latently infected resting CD4 T cells have beendetected in the peripheral blood, gastrointestinal (GI) tract, and lymphnodes of HIV-1-infected individuals and are also likely to exist inother organs containing lymphoid tissue.

Highly active antiretroviral therapy (HAART) enables long-termsuppression of plasma HIV-1 loads in infected persons, but low-levelvirus persists and rebounds following cessation of therapy. DuringHAART, this virus resides in latently infected cells, such as restingCD4 T cells, and in other cell types that may support residual virusreplication. Therapeutic eradication will require elimination of virusfrom all reservoirs.

Burkitt's Lymphoma is a germinal center lymphoma originating and growingwithin the secondary lymphatic system, always associated with a c-Mycactivating chromosomal translocation. It is one of the fastest growingcancers and can double in size every 14-18 hours. BL is an aggressiveB-cell lymphoma found in germinal centers of the spleen and secondarylymphatics. BL is named after Dr. Denis Parsons Burkitt, a surgeon whofirst described the disease in 1958 while working in equatorial Africa(Burket, D., 1958). BL is most commonly found in children living insub-Saharan Africa, with the highest incidence and mortality rates foundin East Africa (Orem, J., et al.). Boys are more susceptible to BL thangirls. Outside of Africa, BL is most likely to occur in people who havea compromised immune system.

Among B-cell malignancies, CLL is the most responsive to ibrutinib, andthus unfortunately ibrutinib is not likely to significantly benefitpeople afflicted with Burkitt's Lymphoma and other germinal centerlymphomas. However, the same result to redistribute B-cell cancers intothe circulation where they are more susceptible to chemotherapy and lessproliferative can be achieved for germinal center lymphomas such asBurkitt's lymphoma with the use of agents that ablate secondarylymphatic germinal centers.

Clinical observations on the ability of a Bruton's Tyrosine Kinaseinhibitor ibrutinib for treatment of chronic lymphocytic leukemia hasdemonstrated that redistribution of CLL cells from the lymphatics intothe bloodstream is a contributing mechanism of action to its benefit inCLL. Circulating CLL cells are not proliferative, with proliferation ofthe clone limited to the lymphatic microenvironment. Therefore,redistribution into the blood stream reduces cancerous proliferation.Similarly, redistribution of ALL from the bone marrow to thebloodstream, has also been reported to enhance sensitivity to standardchemotherapy (Chang B Y, Blood 2013 122: 2412-24).

Glucocorticoids have been reported to have multiple and contradictoryactions on lymphocytes, depending on the dose, the duration of dosingand the species investigated. Glucocorticoids have been investigated aslymphocytosis inducing agents, agents which increase circulatinglymphocyte numbers, since 1943 (for review see Burger et al., 2013),typically with the use of prednisone between 0.5 and 1 mg/kg, whichwould be an equivalent 0.1-0.2 mg/kg dexamethasone dose. High dosemethylprednisone (HDMP) used for refractory CLL, in contrast, does notappear to induce lymphocytosis at the methylprednisone equivalent to the0.5-1.0 mg/kg dose at which prednisone did. Lymphotoxic high-dosesteroids are typically considered to be approximately 100 mg daily ofprednisone equivalent, which would be a dexamethasone equivalent dose of16 mg which is approximately 0.23 to 0.32 mg/kg, and which we havedemonstrated is not an effective preconditioning dose. Dexamethasonedoes not reduce germinal centers in mice until an HED of about 3 mg/kgor greater is administered. Prednisone does not significantly impactspleen weights or germinal centers until used at doses in mice over 2.5mg/kg po daily for 13 weeks (Yan et al., 2015), a human dose which wouldhave unacceptable mineralocorticoid activity as a dose of 30 mgs per day(˜0.48-0.72 mg/kg) is considered a high dose in human lupus patients.

For Burkitt's lymphoma (BL) treatment with standard chemotherapyregimens such as COPADM, prednisone is included in various cyclestypically at 60 mg/m², which converts to 1.62 mg/kg prednisone and anequivalent 0.3 mg/kg dexamethasone dose, which is not an effectivepreconditioning dose. Dexamethasone is also used clinically for thetreatment of B-cell cancers, typically in an oral four-five day 40 mgdaily regimen or 6 mg/m² for 5 days. In some indications such as ALL,dexamethasone is given daily for weeks and can be associated withosteonecrosis, particularly in adolescent boys. Risk of osteonecrosiscan be substantially eliminated by alternate week dosing ofdexamethasone and may be particularly present in ALL because of theasparaginase regimen that is part of the treatment for ALL (Chang B Y,Blood 2013 122: 2412-24).

Epstein-Barr virus (EBV) infection is found in nearly all African BLpatients, and chronic malaria is believed to reduce resistance to EBV,allowing it to take hold. The disease characteristically involves thejaw or other facial bone, distal ileum, cecum, ovaries, kidney, orbreast. Additionally, BL strikes immunocompromised people, such as thosewith HIV.

BL is classified into three main clinical variants: Endemic, Sporadic,and the Immunodeficiency-associated variants, with the Endemic variant(also called the “African variant”) most commonly occurring in childrenliving in malaria endemic regions of the world.

One effect of the present invention can be to ablate germinal centersand/or marginal zones to selectively drive BL and other germinal centercancer cells or marginal zone cancer cells from the germinal centers ormarginal zones into circulation where they can be more easily killedwith chemotherapy or other agents. This could dramatically, safely andcost-effectively advance BL treatment outcomes.

Asthma is a chronic inflammation characterized by an increased number ofCD8+ Type-1 T-lymphocytes and macrophages in the lung tissue andneutrophils in the airway lumen. Lymphocytes, which are markedlydifferent in the two inflammatory conditions, play a crucial role in thepathogenesis of asthma and COPD. There is now overwhelming evidence tosupport a major role for T cells in asthma, in particular theinvolvement of T helper type 2 (Th2) cells in atopic allergic asthma aswell as nonatopic and occupational asthma. There may also be a minorcontribution from T cytotoxic type 2 CD8+ T cells. Several Th2 cytokineshave potential to modulate airway inflammation, in particularinterleukin-13 which induces airway hyperresponsiveness independently ofIgE and eosinophilia in animal models. Asthma and chronic obstructivepulmonary disease (COPD) are two different inflammatory disorders of thelungs which share a common functional abnormality, i.e. airflowlimitation (Baraldo et al., 2007).

In asthma, airflow limitation is largely reversible, eitherspontaneously or with treatment, and does not progress in most cases. Onthe other hand, airflow limitation in COPD is usually progressive andpoorly reversible. In asthma, the chronic inflammation causes anassociated increase in airway responsiveness to a variety of stimuli,leading to recurrent episodes of wheezing, breathlessness, chesttightness and cough, particularly at night and in the early morning.Many cells are involved in the inflammatory response in asthma and,among these, CD4+ Type-2 lymphocytes, mast cells and eosinophils arethought to play a crucial role. In COPD, the poorly reversible airflowlimitation is associated with an abnormal inflammatory response of thelungs to noxious particles or gases. This chronic inflammation ischaracterized by an increased number of CD8+ Type-1 T-lymphocytes andmacrophages in the lung tissue and neutrophils in the airway lumen.Lymphocytes, which are markedly different in the two inflammatoryconditions, play a crucial role in the pathogenesis of asthma and COPD(Baraldo et al., 2007).

Definitions

Definitions used to describe the embodiments of the invention:

Biologic mechanism of lymphodepletion means induction of programmed celldeath via apoptosis or necroptosis or pyroptosis or autophagy oroncosis. Various stimuli can engage a non-apoptotic form of cell deathcalled necroptosis, which occurs when caspases required for apoptosisare inhibited. Pyroptosis is a caspase-dependent form of programmed celldeath that differs in many respects from apoptosis. Unlike apoptosis, itdepends on the activation of caspase-1 or caspase-11 (caspase-5 inhumans). Autophagy is a lysosome-dependent process.

Apoptosis: A form of cell death in which a programmed sequence of eventsleads to the elimination of cells without releasing harmful substancesinto the surrounding area. Apoptosis plays a crucial role in developingand maintaining the health of the body by eliminating old cells,unnecessary cells, and unhealthy cells.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; Band C; A (alone); B (alone); and C (alone).

The term “about’ when referring to a measurable value such as an amountor a temporal duration and the like refers to variations of +/−20% or+/−10%.

Administering” refers to the physical introduction of an agent to asubject, using any of the various methods and delivery systems known tothose skilled in the art. Exemplary routes of administration for theformulations disclosed herein include intravenous, intramuscular,subcutaneous, intraperitoneal, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion, as well as in vivoelectroporation. In some embodiments, the formulation is administeredvia a non-parenteral route, e.g., orally. Other non-parenteral routesinclude a topical, epider-mal or mucosal route of administration, forexample, intranasally, vaginally, rectally, sublingually or topically.

A pharmacologic dose is a dose far in excess of normal levels in thebody.

An “anti-tumor effect” as used herein, refers to a biological effectthat can present as a decrease in tumor volume, a decrease in the numberof tumor cells, a decrease in tumor cell proliferation, a decrease inthe number of metastases, an increase in overall or progression-freesurvival, an increase in life expectancy, or amelioration of variousphysiological symptoms associated with the tumor. An anti-tumor effectcan also refer to the prevention of the occurrence of a tumor, e.g., avaccine.

A therapeutic agent is an agent that enhances the efficacy of cellularimmunotherapies compared to the cellular immunotherapies without saidtherapeutic agent.

The term “autologous” refers to any material derived from the sameindividual to which it is later to be re-introduced, whether theindividual is a human or other animal.

The term “allogeneic” refers to any material derived from one individualwhich is then introduced to another individual of the same species,whether the individual is a human or other animal.

The term dexamethasone (also referred to as Dex) non-exclusively relatesto any formulation whether a liquid solution, liquid suspension, oralsolution, tablet form, tablet form dissolved in a liquid containing theactive ingredient of dexamethasone, injectable form, gel formulation,patch formulation or any formulation containing the active ingredientdexamethasone.

The term glucocorticoid-receptor modulating agents non-exclusivelyrelates to glucocorticoid receptor agonists or glucocorticoid receptormodulators including but not limited to: compound A [CpdA;(2-((4-acetophenyl)-2-chloro-N-methyl)ethylammonium-chloride)] andN-(4-methyl-1-oxo-1H-2,3-benzoxazine-6-yl)-4-(2,3-dihydrobenzofuran-7-yl)-2-hydroxy-2-(trifluoromethyl)-4-methylpentanamide(ZK216348), AL-438, Mapracorat, LGD-5552, RU-24858, Fosdagrocorat,PF-802, Compound 10, MK5932, C108297, LGD5552, and ORG 214007-0.

Immunotoxins are proteins that contain a toxin along with an antibody orgrowth factor that binds specifically to target cells. Immunotoxins arecreated by chemically conjugating an antibody to a whole protein toxin,devoid of its natural binding domain. Immunologic proteins that aresmaller than monoclonal antibodies (MoAbs), like growth factors andcytokines, have also been chemically conjugated and genetically fused toprotein toxins. Toxins used in immunotoxin constructs are derived frombacteria, fungi, and plants, and most function by inhibiting proteinsynthesis. Bacterial toxins commonly used in immunotoxins includeDiphtheria toxin (DT) and the toxin from Pseudomonas exotoxin (PE).Plant toxins utilized in immunotoxins include the A chain of ricin(RTA), and the ribosome inactivating proteins (RIPs) gelonin, pokeweedantiviral protein, and dodecandron. Because it is an enzyme, one toxinmolecule can work on many substrate molecules, having a devastatingeffect on the cell. Toxins such as diphtheria toxin (DT) and Pseudomonasexotoxin (PE) prevent protein synthesis by an effect on elongationfactor 2 (EF-2).

The term systemic injection as used herein non-exclusively relates to aroute of administration that rapidly, within seconds or a few hours,leads to circulating levels of cellular immunotherapies, andnon-exclusively relates to intravenous, intraperitoneally, subcutaneous,via nasal submucosa, lingual, via bronchoscopy, intravenous,intra-arterial, intra-muscular, intro-ocular, intra-striatal,subcutaneous, intradermal, by dermal patch, by skin patch, by patch,into the cerebrospinal fluid, into the portal vein, into the brain, intothe lymphatic system, intra-pleural, retro-orbital, intra-dermal, intothe spleen, intra-lymphatic, among others.

The term ‘site of injection’ as used herein non-exclusively relates tointra-tumor, or intra-organ such as the kidney or liver or pancreas orheart or lung or brain or spleen or eye, intra-muscular, intro-ocular,intra-striatal, intradermal, by dermal patch, by skin patch, by patch,into the cerebrospinal fluid, into the brain, among others.

The term lymphodepletion as used herein non-exclusively relates to thereduction of lymphocyte number in the peripheral blood without causingredistribution of lymphocytes to another organ such as the bone marrow,thymus, lymph nodes, lung or spleen or another organ.

The term lymphoablation as used herein non-exclusively relates toreduction of lymphocyte number in the peripheral blood to below 200 permicroliter, preferably to below 100 per microliter, without causingredistribution of lymphocytes to another organ such as the bone marrow,thymus, lymph nodes, lung or spleen or another organ.

The term cytotoxic lymphodepletion as used herein relates to thereduction of lymphocyte number in the peripheral blood by a mechanism ofADCC, cell-mediated cytotoxicity or direct lysis or cytotoxicelimination of lymphocytes, chemotherapy or radiation.

The antibody-dependent cell-mediated cytotoxicity (ADCC), also referredto as antibody-dependent cellular cytotoxicity, is a mechanism ofcell-mediated immune defense where an effector cell of the immune systemactively lyses a target cell, whose membrane-surface antigens have beenbound by specific antibodies.

The term ‘cellular immunotherapy’, ‘adoptive cellular immunotherapy’,‘adoptive cellular therapy’ (ACT) or cell immunotherapy or cell therapyas used herein non-exclusively relates to treatments that contain a cellused to help the immune system fight diseases or a cell from the immunelineage which directly fights diseases such as cancer, autoimmunediseases and infections with certain viruses. The cellular immunotherapycan be from either an autologous or allogeneic source. In preferredembodiments, the adoptive immunotherapy used in the methods disclosedherein may be an adoptive T cell immunotherapy, i.e. ‘T cell therapy’.

The term preconditioning as used herein relates to the preparation of apatient with a cytotoxic lymphodepleting agent or a non-toxiclymphodepleting agent prior to ACT.

The term immunotherapy, also called biologic therapy, as used hereinnon-exclusively relates to a type of treatment for cancer, autoimmunedisease or infection treatment designed to boost the body's naturaldefenses to fight the cancer, autoimmune disease or infection. It usessubstances either made by the body or in a laboratory to improve orrestore immune system function. The term “immunotherapy” refers to thetreatment of a subject afflicted with, or at risk of contracting orsuffering a recurrence of, a disease by a method comprising inducing,enhancing, suppressing or otherwise modifying an immune response.Examples of immunotherapy include, but are not limited to, T celltherapies. T cell therapy can include adoptive T cell therapy,tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous celltherapy, engineered autologous cell therapy (eACT), and allogeneic Tcell transplantation. However, one of skill in the art would recognizethat the conditioning methods disclosed herein would enhance theeffectiveness of any transplanted T cell therapy. Examples of T celltherapies are described in U.S. Patent Publication Nos. 2014/0154228 and2002/0006409, U.S. Pat. No. 5,728,388, and International Publication No.WO 2008/081035.

The term ‘immune modulation’ as used herein non-exclusively relates to,in cancer, autoimmune disease or infection, a range of treatments aimedat harnessing a patient's immune system to achieve tumour, autoimmunecausing cell or viral control, stabilization, and potential eradicationof disease.

The term immunomodulator as used herein non-exclusively relates to achemical agent (such as dexamethasone) or biologic agent (such asHUMIRA® and rituximab) that modifies the immune response or thefunctioning of the immune system (as by the stimulation of antibodyformation or the inhibition of white blood cell activity). Traditionalimmune modulating drugs that are immunesuppressants non-exclusivelyrelates to glucocorticoids, calcineurin inhibitors, antimetabolites, andalkylating agents. Antimetabolites non-exclusively relates topurineanalogues (e.g., azathioprine and mycophenolate mofetil), and folateantagonists (e.g., methotrexate and dapsone).

Immunesuppressants (also termed immunosuppressants) can be chemical orbiologic agents that can suppress or prevent the immune response. Forinstance, antagonists to CD26 and dexamethasone are immunesuppressants.The NTLAs used in this invention may be NTLA immunesuppressants.

The terms “conditioning” and “pre-conditioning” are used interchangeablyherein and indicate preparing a patient or animal in need of a T celltherapy for a suitable condition. Conditioning as used herein includes,but is not limited to, reducing the number of germinal centers andmarginal zones, reducing the number of endogenous lymphocytes, removinga cytokine sink, increasing a serum level of one or more homeostaticcytokines or pro-inflammatory factors, enhancing an effector function ofT cells administered after the conditioning, enhancing antigenpresenting cell activation and/or availability, or any combinationthereof prior to a T cell therapy.

The term ‘adoptive immunotherapy’ or ‘cellular adoptive immunotherapy’as used herein non-exclusively relates to immune cells that arecollected from a patient (autologous or autogenic) or a donor(allogeneic), either related or unrelated, and grown in the laboratory.This increases the number of immune cells that are able to kill cancercells, autoimmune causing cells or fight infections. These immune cellsare given back to the patient to help the immune system fight disease.This is also called cellular adoptive immunotherapy. The immune cell canbe a T cell and/or other cell of the immune system non-exclusivelyrelating to macrophages, monocytes, dendritic cells, neutrophils,granulocytes, phagocytes, mast cells, basophils, thymocytes, or innatelymphoid cells, or any combination thereof.

The term agonist as used herein non-exclusively relates to any entitythat activates a specific receptor or downstream signaling pathwayessential to mediate the receptor's effect(s). Agonists maynon-exclusively relates to but are not limited to antibodies, antibodyfragments, soluble ligands, small molecules, cyclic peptides,cross-linking agents.

The term antagonist as used herein non-exclusively relates to any entitythat interferes with the binding of a receptor's counter structure(s),or with the activation of a specific receptor or downstream signalingpathway essential to mediate the receptor's effect(s). Antagonists maynon-exclusively relates to but are not limited to antibodies, antibodyfragments, soluble ligands, Fc fusion receptors, chimeric receptors,small molecules, cyclic peptides, peptides.

The term inhibitor as used herein non-exclusively relates to any entitythat diminishes the target effect of a specific receptor. Inhibitors maybe small molecules, antisense agents, nucleic acids including siRNA andmicroRNA.

The term “lymphocyte” as used herein includes natural killer (NK) cells,T cells, or B cells. NK cells are a type of cytotoxic (cell toxic)lymphocyte that represent a major component of the inherent immunesystem. NK cells reject tumors and cells infected by viruses. It worksthrough the process of apoptosis or programmed cell death. They weretermed “natural killers” because they do not require activation in orderto kill cells. T-cells play a major role in cell-mediated-immunity (noantibody involvement). Its T-cell receptors (TCR) differentiatethemselves from other lymphocyte types. The thymus, a specialized organof the immune system, is primarily responsible for the T cell'smaturation. There are six types of T-cells, namely: Helper T-cells(e.g., CD4+ cells), Cytotoxic T-cells (also known as TC, cytotoxic Tlymphocyte, CTL, T-killer cell, cytolytic T cell, CDS+ T-cells or killerT cell), Memory T-cells ((i) stem memory T scM cells, like naive cells,are CD45RO−, CCR 7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+ andIL-7Ra+, but they also express large amounts of CD95, IL-2R˜, CXCR3, andLFA-1, and show numerous functional attributes distinctive of memorycells); (ii) central memory TcM cells express L-selectin and the CCR7,they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory T EMcells, however, do not express L-selectin or CCR 7 but produce effectorcytokines like IFNy and IL-4), Regulatory T-cells (Tregs, suppressor Tcells, or CD4+CD25+ regulatory T cells), Natural Killer T-cells (NKT)and Gamma Delta T-cells. B-cells, on the other hand, play a principalrole in humoral immunity (with antibody involvement). It makesantibodies and antigens and performs the role of antigen presentingcells (APCs) and turns into memory B-cells after activation by antigeninteraction. In manmials, immatureB-cells are formed in the bone marrow,where its name is derived from.

The term “cancer” refers to a disease characterized by the uncontrolledgrowth of aberrant cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. Examples ofvarious cancers are described herein and include but are not limited to,breast cancer, prostate cancer, ovarian cancer, cervical cancer, skincancer, pancreatic cancer, colorectal cancer, renal cancer, livercancer, brain cancer, lymphoma, leukemia, lung cancer and the like. Theterms “tumor” and “cancer” are used interchangeably herein, e.g., bothterms encompass solid and liquid, e.g., diffuse or circulating, tumors.As used herein, the term “cancer” or “tumor” includes premalignant, aswell as malignant cancers and tumors.

The particular cancer can be responsive to chemo- or radiation therapyor the cancer can be refractory. A refractory cancer refers to a cancerthat is not amendable to surgical intervention and the cancer is eitherinitially unresponsive to chemo- or radiation therapy or the cancerbecomes unresponsive over time.

An “anti-tumor effect” as used herein, refers to a biological effectthat can present as a decrease in tumor volume, a decrease in the numberof tumor cells, a decrease in tumor cell proliferation, a decrease inthe number of metastases, an increase in overall or progression-freesurvival, an increase in life expectancy, or amelioration of variousphysiological symptoms associated with the tumor. An anti-tumor effectcan also refer to the prevention of the occurrence of a tumor, e.g., avaccine.

The term “progression-free survival,” which can be abbreviated as PFS,as used herein refers to the time from the treatment date to the date ofdisease progression per the revised IWG Response Criteria for MalignantLymphoma or death from any cause.

“Disease progression” is assessed by measurement of malignant lesions onradiographs or other methods should not be reported as adverse events.Death due to disease progression in the absence of signs and symptomsshould be reported as the primary tumor type (e.g., DLBCL).

The “duration of response,” which can be abbreviated as DOR, as usedherein refers to the period of time between a subject's first objectiveresponse to the date of confirmed disease progression, per the revisedIWG Response Criteria for Malignant Lymphoma, or death.

The term “overall survival,” which can be abbreviated as OS, is definedas the time from the date of treatment to the date of death.

The terms “reducing” and “decreasing” are used interchangeably hereinand indicate any change that is less than the original. “Reducing” and“decreasing” are relative terms, requiring a comparison between pre- andpost-measurements. “Reducing” and “decreasing” include completedepletions.

“Treatment” or “treating” of a subject refers to any type ofintervention or process performed on, or the administration of an activeagent to, the subject with the objective of reversing, alleviating,ameliorating, inhibiting, slowing down or preventing the onset,progression, development, severity or recurrence of a symptom,complication or condition, or biochemical indicia associated with adisease. In one embodiment, “treatment” or “treating” includes a partialremission. In another embodiment, “treatment” or “treating” includes acomplete remission.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of’ refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of’ can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of’ can mean a rangeof up to 20% (i.e., ±20%). For example, about 3 mg can include anynumber between 2.3 mg and 3.6 mg (for 20%). Furthermore, particularlywith respect to biological systems or processes, the terms can mean upto an order of magnitude or up to 5-fold of a value. When particularvalues or compositions are provided in the application and claims,unless otherwise stated, the meaning of “about” or “comprisingessentially of’ should be assumed to be within an acceptable error rangefor that particular value or composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Ranges: Various aspects of the invention are presented in range format.The description in range format is for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, a range from3 to 12 includes 3.1, 3.2, 3.3 etc.

Autoimmune disorders and other diseases that are mediated by lymphocytesand which are in need of treatments that are simpler and less costlythan HSCT are related to, but not limited by the following list;allergies, asthma, residual HIV, residual malaria, germinal centerlymphomas such as Burkitts Lymphoma and Diffuse Large B cell Lymphoma,marginal zone lymphoma graft versus host disease (GvHD),steroid-resistant GvHD, Achalasia, Addison's disease, Adult Still'sdisease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome,Autoimmune angioedema, Autoimmune dysautonomia, Autoimmuneencephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease(AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmuneorchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmuneurticaria, Axonal & neuronal neuropathy (AMAN), Baló disease, Behcet'sdisease, Benign mucosal pemphigoid, Bullous pemphigoid, Castlemandisease (CD), Celiac disease, Chagas disease, Chronic inflammatorydemyelinating polyneuropathy (CIDP), Chronic recurrent multifocalosteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or EosinophilicGranulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Coldagglutinin disease, Congenital heart block, Coxsackie myocarditis, CRESTsyndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis,Devic's disease (neuromyelitis optic), Discoid lupus, Dressler'ssyndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilicfasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evanssyndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Giant cell myocarditis, Glomerulonephritis,Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoidgestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa),Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosingdisease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis(IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus, Lyme disease chronic, Meniere's disease, Microscopic polyangiitis(MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB,Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, NeonatalLupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid,Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplasticcerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria(PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis),Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritisnodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cellaplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Relapsing polychondritis,Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis,Scleroderma, Sjögren's syndrome, Sperm & testicular autoimmunity, Stiffperson syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac'ssyndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporalarteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes,Ulcerative colitis (UC), Undifferentiated connective tissue disease(UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease

In certain embodiments of this invention one might want to excludediseases such as allergies, asthma, residual HIV, residual malaria,germinal center lymphomas such as Burkitts Lymphoma and Diffuse Large Bcell Lymphoma, marginal zone lymphoma, graft versus host disease (GvHD),steroid-resistant GvHD, Achalasia, Addison's disease, Adult Still'sdisease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome,Autoimmune angioedema, Autoimmune dysautonomia, Autoimmuneencephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease(AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmuneorchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmuneurticaria, Axonal & neuronal neuropathy (AMAN), Baló disease, Behcet'sdisease, Benign mucosal pemphigoid, Bullous pemphigoid, Castlemandisease (CD), Celiac disease, Chagas disease, Chronic inflammatorydemyelinating polyneuropathy (CIDP), Chronic recurrent multifocalosteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or EosinophilicGranulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Coldagglutinin disease, Congenital heart block, Coxsackie myocarditis, CRESTsyndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis,Devic's disease (neuromyelitis optic), Discoid lupus, Dressler'ssyndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilicfasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evanssyndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Giant cell myocarditis, Glomerulonephritis,Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoidgestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa),Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosingdisease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis(IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus, Lyme disease chronic, Meniere's disease, Microscopic polyangiitis(MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB,Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, NeonatalLupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid,Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplasticcerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria(PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis),Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritisnodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cellaplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Relapsing polychondritis,Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis,Scleroderma, Sjögren's syndrome, Sperm & testicular autoimmunity, Stiffperson syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac'ssyndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporalarteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes,Ulcerative colitis (UC), Undifferentiated connective tissue disease(UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease.

Further Discussion of the Immune Setting of the Invention

The spleen contains both a white pulp and a red pulp. The red pulp ofthe spleen holds macrophages that normally filter and remove senescentor defective red blood cells (RBCs) and antibody-coated bacteria or redblood cells from the circulation. The white pulp of the spleen containsthe lymphoid compartments and is crucial for immune surveillance andresponse: it synthesizes antibodies against invading pathogens andreleases platelets and neutrophils in response to bleeding or infection.During development the spleen is believed to have multiple rolesincluding being the first site of hematopoiesis (at six weeks ofgestation). Preclinical and clinical trials have demonstrated thatwithout cytotoxic chemotherapy preconditioning, cellular immunotherapiesare cleared from the circulation, largely within one hour afteradministration, and accumulate in the spleen. The cytotoxic chemotherapypreconditioning must be immediate to administration of the cellularimmunotherapies in order to maintain the cellular immunotherapies in thecirculation, typically 48 hours before administration of the cellularimmunotherapies. When cytotoxic chemotherapy preconditioning is given 4weeks before or at a pretreatment time which allows bone marrowrecovery, it is not effective to keep the cellular immunotherapies inthe circulation Ritchie D S et al. Mol Ther. November; 21(11): 2122-9(2013)

The periarterial lymphoid sheaths (PALS) of the white pulp of the spleenare populated mainly by T cells, while the lymphoid portions arepopulated mainly by B cells. Germinal centers (GC) are sites withinlymph nodes or lymph nodules in peripheral lymph tissues, and in thewhite pulp of the spleen where intense mature B lymphocytes, otherwiseknown as Centrocytes rapidly proliferate, differentiate, mutate throughsomatic hypermutation and class switch during antibody responses.Germinal centers are an important part of the B-cell humoral immuneresponse. They develop dynamically after the activation of B-cells byT-dependent antigen. Histologically, the GCs describe microscopicallydistinguishable parts in lymphoid tissues. Activated B-cells migratefrom the primary focus into the primary follicles follicular system andbegin monoclonal expansion in the environment of follicular dendriticcells (FDC).

After several days of expansion the B cells mutate theirantibody-encoding DNA and thus generate a diversity of clones in thegerminal center. This involves random substitutions, deletions andinsertions due to somatic hypermutation. Upon some unidentified stimulusfrom the FDC, the maturing B cells (Centroblasts) migrate from the darkzone to the light zone and start to expose their antibody to theirsurface and in this stage are referred to as Centrocytes. TheCentrocytes are in a state of activated apoptosis and compete forsurvival signals from FDCs that present the antigen. This rescue processis believed to be dependent on the affinity of the antibody to theantigen. The functional B-cells have then to interact with helper Tcells to get final differentiation signals. This also involves isotypeswitching for example from IgM to IgG. The interaction with T cells isbelieved to prevent the generation of autoreactive antibodies. The Bcells become either a plasma cell spreading antibodies or a memory Bcell that will be activated in subsequent contacts with the sameantigen. They may also restart the whole process of proliferation,mutation and selection according to the recycling hypothesis.

The B cells contained within the white pulp region of the spleen can befurther divided into specific areas, identified by staining withspecific molecular markers. The marginal zone of the spleen containsnoncirculating mature B cells that border on the white pulp creating aseparation between the white and the red pulp and express high levels ofCD21 and IgM and CD24 and CD79a, and measurable levels of CD9 and CD22.The mantle zone surrounds normal germinal center follicles and expressesCD21, CD23 and CD38. The follicular zone is contained within thegerminal centers and expresses high levels of IgD and CD23, intermediatelevels of CD21 and CD24, and can also be identified by PNA staining. Thegerminal center is best distinguished by PNA binding and expresseshigher levels of CD54 than the follicular zone. Germinal centers have aspecial population of helper T cells that seem to distribute evenly inall germinal centers. Germinal centers are traditionally associated withimmune responses that require T helper cells, although this is notabsolute. Germinal centers are where hypervariable gene mutation occursand high affinity IgG producing B cells are generated. Active germinalcenters have tangible macrophages and CD21 expressing dendritic cells.Follicular centers can also be identified by the expression of CD45R(B220) (Cytotoxicologic Pathology, 35:366-375, 2007). CD45R follicularcenters are found surrounding germinal centers expressing Bc16 and Bc12.BioEssays 29:166-177, 2007; Cytotoxicol Pathol 34(5): 648-655, (2006)]

The response to pathogens or cancer cells is orchestrated by the complexinteractions and activities of the large number of diverse cell typesinvolved in the immune response. The innate immune response is the firstline of defense and occurs soon after pathogen exposure. It is carriedout by phagocytic cells such as neutrophils and macrophages,cytocytotoxic natural killer (NK) cells, and granulocytes. Thesubsequent adaptive immune response elicits antigen-specific defensemechanisms and may take days to develop. Cell types with critical rolesin adaptive immunity are antigen-presenting cells including macrophagesand dendritic cells. Antigen-dependent stimulation of various cell typesincluding T cell subsets, B cells, and macrophages all play criticalroles in host defense. Immune cells non-exclusively relates to: B Cells,Dendritic Cells, Granulocytes, Innate Lymphoid Cells (ILCs),Megakaryocytes, Monocytes/Macrophages, Myeloid-derived Suppressor Cells(MDSC), Natural Killer (NK) Cells, Platelets, Red Blood Cells (RBCs), TCells, Thymocytes.

Zwang et al (2014) have shown that, following lymphodepletion,lymphocytes repopulate the immune space both through enhancedthymopoiesis and proliferation of residual non-depleted peripherallymphocytes. The term homeostatic proliferation (alternativelyhomeostatic expansion or lymphopenia-induced proliferation) refers tothe latter process. Homeostatic proliferation is especially relevant toreconstitution of the lymphocyte compartment following immunodepletiontherapy in transplantation. Repopulating lymphocytes can skew toward aneffector memory type capable of inducing graft rejection, autoimmunity,or, in the case of allogeneic bone marrow transplantation, graft versushost disease.

Two immune-depleting agents, alemtuzumab and rabbit antithymocyteglobulin, have been well-characterized in their abilities to induce aneffector-memory phenotype in repopulating lymphocytes.

Early studies of homeostatic proliferation showed that T cells survivinglymphodepletion divided, developed memory phenotype and function, andthen acted in a dominant fashion to render animals resistant to cardiacor renal allograft tolerance via costimulatory blockade.1,2 In line withthese findings, recent studies have shown that lymphopenia itself isenough to break stable costimulatory blockade-based peripheraltolerance.3 In a mouse model of MHC-mimatched cardiac transplantation,lymphopenia (achieved either by irradiation or anti-CD4+/CD8+ monoclonalantibodies) induced acute T and B cell-mediated rejection, accompaniedby a T cell shift toward a CD44hi effector-memory (EM) phenotype and theappearance of donor-specific antibodies. The process of homeostaticproliferation can be divided into “slow” (one cell division per 24-36hours) or “fast” (one division per 6-8 h) kinetics. While slowproliferation occurs in response to a “sensing of empty space”, rapidproliferation is primarily a gut antigen-driven process.4 Slowhomeostatic proliferation predominates in homeostatic proliferationfollowing lymphodepletion in mouse models. Furthermore, both T and Bcells can undergo homeostatic proliferation.

Alemtuzumab (anti-CD52) is a potent lymphocyte depletional agent thathas been used as induction therapy for transplantation and for treatmentof multiple sclerosis. CD4+ cells and, to a lesser extent, naïve CD8+cells, are most susceptible to alemtuzumab-inducedlymphodepletion.5,6,7,8 A larger population of naïve T cells may remainundeleted, however, as peripheral lymph nodes may be a reservoir forthese cells following alemtuzumab induction.9 Alemtuzumab therapy leadsto skewing toward memory CD4+ and CD8+ phenotypes in renal transplantrecipients; those with evidence of rejection (by biopsy, new ordonor-specific antibodies) following alemtuzumab therapy have anincreased proportion of CD8+ effector memory cells (CD45RO−CD62L−).10These same patients further have decreased frequencies of regulatory Tcells (Tregs) among CD4+ cells. While other work, in contrast, hassuggested an increased frequency of Foxp3+ cells following alemtuzumabinduction.11 It is possible that in this instance Foxp3 expression maybe only a transient marker of T cell activation.12,13, 14 Among patientswith multiple sclerosis, homeostatic proliferation following alemtuzumabtherapy leads to recovery of a highly activated, proliferative,oligoclonal, and memory-like population of CD4+ and CD8+ cells.15 Inparticular, the CD8 pool is dominated by a terminally-differentiated,effector memory CD28−CD57+CD8 population expressing perforin andGranzyme B. Such as population is known to be associated withautoimmunity, and indeed in this study of 87 patients, two thirdsdeveloped (primarily thyroid) autoimmunity.

Recent examination of the kinetics of lymphocyte depletion followingrATG given as induction therapy in renal transplantation found that rATGdurably depletes the T cell compartment to counts below 250 CD3+cells/uL at six months, compared to minimal T cell depletion followingbasiliximab or no induction therapy.19 In contrast to prior studies,this recent investigation found no increase in thymopoiesis (i.e., CD31+cells among CD4+ or CD8+ cells) one month following rATG induction.Rather, peripheral cytokine-mediated signaling by IL-7 and IL-15 viaStatS increased in the first month following rATG therapy, particularlyamong memory T cell subsets. These studies indicate that T cell recoveryfollowing ATG comes from peripheral T cell pools rather than heightenedthymopoiesis.

In humans, unlike mice, the majority of proliferating T cells derivesfrom the periphery rather than the thymus.20 Therefore, peripheralcytokine signaling is essential to maintain the lymphoreplete state andrepopulate the T cell compartment in lymphopenia. IL-7 is the primarycytokine responsible for T cell homeostatic proliferation. In youngthymectomized and elderly adults, circulating IL-7 levels are higherthan those of healthy controls.21 IL-7 in these patients with low or nothymic function appears to stimulate T cell proliferation via STATSsignaling. IL-7 itself has been described as a “rheostat” to maintainthe T cell compartment.22 In lymphopenia, excess IL-7 stimulates T cellproliferation. Proliferating T cells consume IL-7, and levels fall tothe basal state as the T cell compartment repopulates. This mechanismprevents excess proliferation and preserves T cell homeostasis. A recentstudy found that IL-7-induced proliferation requires intermittent(rather than continuous) signaling and that TCR engagement provides thisinterruption.23 T cells with inadequate affinity for peripheral (self)TCR ligands die following prolonged IL-7 signaling; this mechanismmaintains a population of T cells with appropriate affinity for selfligands. In addition to IL-7, IL-15 signaling is important for CD8+ Tcell survival and proliferation.24,25,26 While IL-15 enhanceshomeostatic proliferation of memory CD8+ cells, IL-15 alone is notenough for homeostatic proliferation of naïve CD8 T cells. 27 In naïveCD8+ cells, MHC I engagement is also necessary for homeostaticproliferation.28 Emerging data show that memory CD4+ may also beresponsive to IL-15.29,30,31 Finally, TGB-β may attenuate IL-15signaling and act as a brake on homeostatic proliferation-drivenautoimmunity.32,33, 34,35,36,37

The protein tyrosine phosphatase gene product PTPN2, which dampens TCRsignaling in CD4+ and CD8+ cells, is implicated in humanautoimmunity.38,39 T cell knockout of PTPN2 in a mouse model resulted inmore rapid lymphopenia-induced CD8+ proliferation compared to controlanimals. Adoptive transfer of PTPN2-deleted CD8+ cells into congenichosts resulted in effector/memory differentiation and autoimmunitycompared to adoptive transfer of control CD8+ cells.40 This response wasIL-7-independent. miRNA-181a enhances TCR signaling, in part bysuppressing expression of other protein phosphatases.41 Thus, miRNA-181or another miRNA might inhibit PTPN2 expression and thereby dampenlymphopenia-induced proliferation. It has been suggested thattranscription factors may regulate the ability of hematopoietic stemcells to repopulate the lymphocyte compartment. For example, Hoxb4signaling may promote a hematopoietic stem cell CD4+ central memory(CD44hiCD62L+) phenotype in response to lymphopenia.42 In competitiveadoptive transfer experiments, Hoxb4− overexpressing central memorycells contributed less than wild-type central memory cells toreconstitution of lymphoid organs. Finally, the integrin CD18(lymphocyte function-associated antigen-1, or LFA-1) functions in naïveT cell trafficking between the gut and secondary lymphoid organs43,44and is implicated in gut autoimmunity.45 Adoptive transfer ofCD4+CD18−/− cells into Rag−/− hosts has shown the requirement of CD18both for fast and slow lymphopenia-induced proliferation.46 The abovestudies have illustrated the importance of non-cytokine regulators ofhomeostatic proliferation that skew toward an effector memory phenotypein homeostatic proliferation.

Another potential approach to overcoming homeostatic proliferation as abarrier to transplantation is to delete potentially pathologic CD8+cells specifically in transplant recipients. Yamada et al employed thisapproach with the use of anti-CD8 mAbs at the time of lymphodepletion ina mixed chimerism model of MHC mismatched renal transplantation innonhuman primates; 54 their findings of decreased Tmem responses inCD8-depleted animals are encouraging. The same group subsequentlystudied alefacept, a fusion protein of the extracellular CD2-bindingportion of the human leukocyte function antigen-3 (LFA-3) adhesionmolecule.55 This agent is thought to interrupt cytotoxic effector memoryT cell proliferation by blocking the interaction between effector-memoryCD2+ cells and LFA-3. Alefacept therapy for psoriasis preferentiallydepleted CD4+CD45RO+ effector memory cells, which correlated withclinical improvement in skin lesions.56 Alefacept preferentially andreversibly depleted CD8+ effector memory (CD28−CD95+) cells in anonhuman primate transplantation mode157; CD28− cells in this model wereCD2hi, helping to explain alefacept's ability to preferentially depleteCD8+ cells.

Post-transplant cyclophosphamide administration is an attractiveapproach to prevent GVHD by depleting alloreactive CD8+ cells that mightotherwise survive induction therapy.58,59 Recent data suggest thatpost-transplant cyclophosphamide administration primarily targetsrapidly-dividing allo-specific cells, relatively sparing naïve cellsessential to maintenance of immunocompetence following HSCT.60CD4+Foxp3+Tregs appear resistant to cyclophosphamide and recover quicklyfollowing cyclophosphamide induction for allogeneic bone marrowtransplantation.61 Sparing of Tregs may partly underlie the mechanism bywhich cyclophosphamide prevents GVHD.

Thangavelu et al., (2005) demonstrated prolonged, profoundCD4+T-lymphopenia in rheumatoid arthritis (RA) patients followinglymphocyte-depleting therapy. Poor reconstitution could result eitherfrom reduced de novo T-cell production through the thymus or from poorperipheral expansion of residual T-cells. Interleukin-7 (IL-7) is knownto stimulate the thymus to produce new T-cells and to allow circulatingmature T-cells to expand, thereby playing a critical role in T-cellhomeostasis. In the present study we demonstrated reduced levels ofcirculating IL-7 in a cross-section of RA patients. IL-7 production bybone marrow stromal cell cultures was also compromised in RA. Toinvestigate whether such an IL-7 deficiency could account for theprolonged lymphopenia observed in RA following therapeuticlymphodepletion, we compared RA patients and patients with solid cancerstreated with high-dose chemotherapy and autologous progenitor cellrescue. Chemotherapy rendered all patients similarly lymphopenic, butthis was sustained in RA patients at 12 months, as compared with thereconstitution that occurred in cancer patients by 3-4 months. Bothcohorts produced naive T-cells containing T-cell receptor excisioncircles. The main distinguishing feature between the groups was afailure to expand peripheral T-cells in RA, particularly memory cellsduring the first 3 months after treatment. Most importantly, there wasno increase in serum IL-7 levels in RA, as compared with a fourfold risein non-RA control individuals at the time of lymphopenia. Our datatherefore suggest that RA patients are relatively IL-7 deficient andthat this deficiency is likely to be an important contributing factor topoor early T-cell reconstitution in RA following therapeuticlymphodepletion. Furthermore, in RA patients with stable, wellcontrolled disease, IL-7 levels were positively correlated with theT-cell receptor excision circle content of CD4+ T-cells, demonstrating adirect effect of IL-7 on thymic activity in this cohort.

EXAMPLES

The following examples demonstrate that high dose glucocorticoidreceptor agonists can cause near complete lymphodepletion of peripheralblood lymphocytes as well as reduce the number of germinal centers inlymphoid organs and deplete thymus lymphocytes. These effects areachieved without substantially affecting cell counts of neutrophils,platelets, RBCs and stem cells (both HSCs and MSCs).

These examples also show that this lymphodepletion profile of high dosesof glucocorticoid agonists is similar to that of standard chemotherapyregimens (based on Cyclophosphamide (Cy) and Fludarabine (Flu)), butdoes not elicit associated weight loss (a general measure of toxicity ofsuch chemotherapeutic regimens).

High doses of glucocorticoid agonists thus represent a non-myeloablativeregimen that can produce “immunologic reset” with efficacy comparable tochemotherapy but without associated toxicity. Accordingly, high doseglucocorticoid receptor agonists represent a promising therapy for usein the treatment of diseases mediated by immune cells such aslymphocytes.

Example 1—Immunosuppressant Reduction of Secondary and Primary LymphaticSites

Acute high dose dexamethasone may also be referred to herein as Dex,AugmenStem™, PlenaStem™ or AVM0703.

For mice, male mice were intraperitoneally injected with dexamethasonesodium phosphate for 114.6 mg/kg dexamethasone base (HED 9.32 mg/kg) day0 and were sacrificed 96 hours after the dexamethasone injection. Themice were sacrificed by exsanguination and then residual blood cellsflushed out with 5 U heparin/ml PBS via retrograde flush into thethoracic jugular vein. The spleens were removed, weighed wet, and thenfixed in 10% formalin. Subsequently the spleens were sectioned viaproprietary methods and then incubated with FITC-PNA at 4 degC for 24hours, washed, placed on slides and immunofluorescent images werecaptured. Metamorph software was used to quantify the immunofluorescentsignal. Sample images and the results, normalized to spleen area, areshown in FIG. 1.

Control mice have significant FITC-PNA immunofluorescence, while micewho were injected with dexamethasone sodium phosphate have almost noimmunofluorescent signal. FITC-PNA labels germinal centers, whichnon-exclusively relates to the spleen and lymph nodes. This exampledemonstrates the ability of high dose dexamethasone to reduce the numberof germinal centers (GCs) in lymphoid organs, which could eliminateautoreactive immunologic memory. Reducing the number of germinal centersin lymphoid organs can also force cancer cells (for example germinalcenter lymphomas) or residual HIV infected T cells, which bind to nichesin these centers, into the circulation where they can be eliminated bythe immune system or standard therapies.

FIG. 2 shows the dose response of acute high dose dexamethasone (in HED)effect on germinal center number in spleens of mice. Germinal centerreduction is apparent at HED 6 mg/kg but not significantly reduced untilHED of 9 and 12 mg/kg doses.

For rat, dexamethasone HED between 3.23, 6.45 and 12.9 mg/kg (rat doses20, 40 and 80 mg/kg) was administered (IV or PO) to determine GC andmarginal zone inhibition 48 hours later. In the rat, the HED Dex dose of12.9 mg/kg maximally inhibited both GC and marginal zone number and areaas shown in FIG. 3 and FIG. 4. Formalin-fixed spleens werecross-sectioned in 5 pieces, trimmed and embedded in paraffin, sectionedand stained with hematoxylin and eosin (H&E). Measurements of theperiarteriolar lymphoid sheath (PAL) diameter and the width of themarginal zone (MZ) in areas of white pulp that had PAL with the greatestdiameter were measured using an ocular micrometer. BCL-6immunohistochemical staining in rat spleens was evaluated to determineGC area using automated image analysis methods.

Acute high dose dexamethasone also reduces thymic mass and volume (FIG.5). For mice, male mice were orally administered vehicle ordexamethasone sodium phosphate for 3, 6, 9 and 12 mg/kg HEDdexamethasone base day 0 and were sacrificed 48 hours after thedexamethasone treatment. The mice were sacrificed by exsanguination andthen residual blood cells flushed out with 5 U heparin/ml PBS viaretrograde flush into the thoracic jugular vein. The thymus from eachmouse were removed, weighed wet, graphed as thymus weight/body weight.

Example 2—Immunosuppressant Lymphodepletion in Mice and Rats 24-48 Hoursafter Acute Administration of Dexamethasone, with Neutrophil, RBC,Platelet and Stem Cell Sparing Properties

Preliminary dose escalation studies performed in naïve mouse and ratmodels showed that administration of high-dose dexamethasone results incomplete lymphodepletion (FIG. 11, right side). High-doses ofdexamethasone were able to induce ˜98% reduction in CD4+, CD8+, Tregsand B cells population measured 48 hours after administration,supporting rapid ablation of autoimmune pathophysiologic substrates.Early stage validation showed that acute high dose dexamethasone has 2-3hour half-life by pharmacokinetic and a pharmacodynamics half-life of4-5 days, which exclude prolonged immune suppression. In addition, oraldosing of acute high dose dexamethasone has comparable effects to IVdosing, which supports the use of acute high dose dexamethasone as asingle oral treatment.

As shown in FIG. 6, IV or PO administration of dexamethasone at 20 (3.2HED), 40 (6.5 HED) or 80 (12.9 HED) mg/kg to male Lewis rats weighing250-300 grams significantly reduced lymphocyte count at all dosescompared to Placebo 48 hours after administration. In contrast, as shownin FIG. 7, neutrophils were not reduced by acute high dosedexamethasone. Neutrophil number are actually increased by all doses ofdexamethasone, likely via a demargination effect. RBCs, platelets, Hct,HgB were not affected by the dexamethasone treatment.

Oral acute administration of dexamethasone to C57B1 male mice at HED of3 mg/kg (n=4), HED 6 mg/kg (n=6), 9 mg/kg (n=4), 12 mg/kg (n=4), 15mg/kg (n=4) or 17.5 mg/kg (n=4) compared to placebo (n=7) reduced CD3+Tlymphocytes by 65% and CD4+T lymphocytes by 75% (FIG. 8), reduced CD8+Tlymphocytes by 56% and Tregs by 78% (FIG. 9), reduced natural killercells (NK) by 87% and B lymphocytes by 83% (FIG. 10), reduced absolutelymphocyte count by 84% but spared neutrophils (FIG. 11), RBCs (FIG. 12)and platelets (FIG. 13). Blood was drawn for complete blood chemistry(CBC) and flow cytometry 24 to 48 hours after dexamethasoneadministration by oral gavage. At HED doses greater than 12 mg/kg almostcomplete lymphoabalation was observed in normal mice. In tumor bearingmice a near complete lymphoablating dose will be HED greater than 6mg/kg.

Acute high dose dexamethasone activates receptor-mediated apoptosis viathe caspase pathway and lympho-depletes or lympho-ablates alllymphocytes depending on the dose used. As expected from itsreceptor-mediated mode of action, dexamethasone induces lymphodepletionsparing neutrophils, platelets, and Red Blood Cells (RBCs) due to thelack of or due to different glucocorticoid receptors on these cells. Atendency to elevate neutrophil counts above placebo in both peripheralblood and bone marrow was observed with high doses of dexamethasone,supporting possible protection against infections during lymphodepletiontreatments.

Remarkably, high doses of dexamethasone did not significantly alter theamount of hematopoietic stem cells in mice (FIG. 13). Thenon-myeloablative regimen represented by acute high dose dexamethasonecould, therefore, eliminate the need for transfusions of stem cells forhematopoietic recovery after immune-reset.

Example 3—Immunosuppressant Lymphodepletion in Humans 36-48 Hours afterAcute Administration of Dexamethasone, with Neutrophil, RBC, Plateletand Stem Cell Sparing Properties

Oral acute administration of 3 mg/kg dexamethasone base equivalent (alldoses given are dexamethasone base equivalent in these examples) to fourhuman patients, three with knee osteoarthritis and one with aorticaneurysm, was administered. Blood was drawn before drug treatment and 48hours post-treatment for CBC analysis and flow cytometry to determinelymphocyte and other blood cell populations. Serum was analyzed forcytokine levels. For one patient, pre-treatment CBCs were not drawn andthus normalized flow cytometry data is shown for only 3 patients. Byun-normalized flow cytometry data only 2 of the 4 patients responded tothe dexamethasone with lymphodepletion (FIGS. 14, 15, and 16), while 2of 4 patients showed a lymphocytosis response in CD3 and CD4 lymphocytesand 1 of 4 patients showed a lymphocytosis response in CD8, Blymphocytes and NK cells, to this dose of dexamethasone. 3 of 4 patientsshowed elevated levels of IL-2 and 4 of 4 showed elevated levels ofIL-15 48 hours after acute oral dexamethasone base (3 mg/kg) (FIG. 17).IL-6, a cytokine known to be the primary driver of potentially fatalcytokine release syndrome (CRS) was not elevated in any patient. Basedon the lymphocytosis response observed in 2 of 4 non cancer patients atthe 3 mg/kg dose, preferred lymphodepleting doses will be 3 mg/kg orhigher based on the increased sensitivity of tumor bearing mice todexamethasone where the lowest lethal dose was HED 43 mg/kg in tumorbearing mice compared to HED 114 mg/kg in healthy mice (ScorzaBarcellona, 1984).

Bone marrow was drawn 48 hours after dexamethasone administration andmesenchymal stem cell (MSC) number was determined by colony-formingassay fibroblast (CFU-F). Oral administration of dexamethasone base 3mg/kg increased ileac crest bone marrow (BM) MSC almost two fold (FIG.18). Trilineage differentiation capacity of the BM MSC was alsodetermined in a study in horses. A 6 mg/kg HED doubled sternal BM MSCstem cell number 48 hours after a one hour IV infusion administration tohorses, but did not alter trilineage differentiation capacity of the MSCtowards osteocytes, chondrocytes or adipocytes.

Example 4—Comparison of Acute 12 mg/kg and 17.5 mg/kg Dexamethasone BaseHED to a Standard Cy (Cyclophosphamide) Flu (Fludarabine) ChemotherapyRegimen

Dexamethasone base was administered by oral gavage to adult male mice at12 mg/kg or 17.5 mg/kg HED on day −2. To another group of mice Cy wasadministered IP at 166 mg/kg (HED 500 mg/m2) on day −5 and day −4 andFludarabine 10 mg/kg (HED 30 mg/m2) on days −5, −4, −3, −2. To a thirdgroup of mice Cy was administered IP at 166 mg/kg (HED 500 mg/m2) on day−5 and Fludarabine 10 mg/kg (HED 30 mg/m2) on days −5, then 12 mg/kg or17.5 mg/kg HED dexamethasone base was administered orally on day −2. CBCand flow cytometry results are shown in FIGS. 19-24, and body weightsare shown in FIG. 25.

Dexamethasone base 12 mg/kg or 17.5 mg/kg HED given between 12-72 hoursbefore blood draw leads to a comparable lymphodepletion profile comparedto standard 2 day Cy with 4 day Flu, as does the combination of a singleCy on day −5 and a single Flu on day −5 with 12 mg/kg dexamethasone HEDon day −2 (FIG. 23). The single Cy and single Flu dose can beadministered on day −6, day −4, or day-3 with equal effect. Thelymphodepletion profile of dexamethasone alone may be preferable becauseabsolute lymphocytes are not depleted as dramatically as with CyFlu, andthe degree of lymphodepletion may be related to neuroedema when adoptivecell therapy is given after CyFlu.

The standard repeat CyFlu regimen significantly reduced body weight as ageneral measure of toxicity, while 12 mg/kg or 17.5 mg/kg dexamethasonebase HED did not impact body weight. The combination of one Cy and oneFlu dose on day −5 with 12 mg/kg dexamethasone HED impacted body weightsignificantly less than the standard CyFlu regimen, while thecombination of one Cy and one Flu dose on day −5 with 17.5 mg/kgdexamethasone HED did not impact body weight (FIG. 25). Thisdemonstrates that acute high dose dexamethasone has a lymphodepletionprofile equivalent to standard chemotherapy based on Cyclophosphamide(Cy) and Fludarabine (Flu) but with no associated weight loss,confirming the safety of the dexamethasone formulation compared tochemotherapy.

Additionally, in a double-blind controlled horse trial with acute highdose dexamethasone, no adverse side effects were observed for out to 70days.

Data collected to date suggest that acute high dose dexamethasonepresents with a safety profile consistent with that of approved DSPproducts. The proposed doses of acute high dose dexamethasone (HED 15-26mg/kg) are equivalent or less than the cumulative doses of DSP that areused safely and effectively for pulse therapy daily for up to 5 days fora variety of conditions, and DSP has been well tolerated in physicianinitiated high-dose pulse therapy clinical use (Han et al, 2014; Annaneet al, 2004; Ayache et al, 2014). A preliminary study performed on asmall number of human osteoarthritis patients revealed that acute highdose dexamethasone elevates levels of plasma IL-2 and IL-15 cytokines,without affecting the concentration of pro-inflammatory cytokines (e.g.IL-6), as seen after chemotherapy regimens (U.S. Pat. No. 9,855,298B2).A full analysis of clinical chemistries in mice treated with acute highdose dexamethasone at increasing HED doses (6-12 mg/kg) showed thatacute oral doses are safe and do not elevate clinical chemistry levelsout of normal range including cholesterol and total protein. Moreover,while chronic low doses of DSP have been shown to cause undesirable sideeffects, including weight gain and glucose increase (Ferris & Kahn,2012), the glucose level after acute high dose dexamethasone has beenfound not elevated over the normal range. Altogether, thelymphodepleting activity of acute high dose dexamethasone and its safeprofile strongly support its use as immunologic reset treatment forautoimmune diseases with efficacy comparable to chemotherapy.

Other standard chemotherapeutic regimens that can be given as a singledose(s) on day −1 or day −2 or day −3 or day −4 or day −5 and becombined with Dexamethasone between about 3 to about 12 mg/kg on day −2include: Cy 120 mg/kg and Flu 75 mg/m2; 30 mg/m2 flu and 50 mg/kg Cy and200 cGy TBI; Cy 1500 mg/m2 and Bendamustine 120 mg/m2; Cy between about300 mg/m2 and about 2300 mg/m2; Flu between about 10 mg/m2 and about 900mg/m2; Cy 600 mg/m2 and Flu 30 mg/m2; Busulfan and Melphalan and Flu;Busulfan (dose adjusted according to weight) and Thiotepa (10 mg/kg) andFludarabine (160 mg/m2); Flu 30 mg/m2 and Cy 300 mg/m2 and Mensa 300mg/m2; Flu 30 mg/m2 and Cy 60 mg/m2 and Alemtuzumab 0.2 mg/kg.

Example 5—Treatment of Patients with Autoimmune Diseases

A patient with an autoimmune disease such as, but not limited to: SLE,psoriasis, rheumatoid arthritis, sporiatic arthritis, type I diabetes,multiple sclerosis, Sjogren's Syndrome, scleroderma, Grave's Disease,Hashimoto's thyroiditis, Celiac Disease, Addison's Disease, MyastheniaGravis, Autoimmune hepatitis, Antiphospholipid syndrome, biliarycholangitis, can be treated with a glucocorticoid immune suppressant, orwith dexamethasone dose. Acute high dose dexamethasone (as base) dosesrange from about 15 mg/kg to about 26 mg/kg, with doses between about 15mg/kg and about 21 mg/kg being preferred.

B lymphocyte numbers are reduced by greater than 90% with acute highdose dexamethasone doses, and as memory B cells make up approximately50% of the B cell compartment in people over age 20, memory B cellpopulations are also reduced by greater than 90%. The patient'sautoimmune attacking B cells have apoptosed and the patient ceases tohave active self-immune attacks. The patient's physical symptoms areimproved or eliminated. Remission from the autoimmune disease lastsindefinitely in most patients, however, should the patient relapse thena repeat dose of the glucocorticoid immune suppressant, dexamethasonedoses, or antagonist to CD26 can be administered. Repeat treatments canoccur as often as once per month if necessary, but preferably not morethan one a year, and most preferably not more than once every 5 years.

Example 6—Treatment of Residual HIV

A patient with residual HIV is treated with glucocorticoid immunesuppressant, or with dexamethasone. Acute high dose dexamethasone (asbase) doses range from about 15 mg/kg to about 26 mg/kg, with dosesbetween about 15 mg/kg and about 21 mg/kg being preferred. The treatmenteliminates the niches in the spleen where HIV hides and sends theinfected T cells into the circulation where they can be killed bystandard HIV therapies that include anti-retroviral drugs, including butnot limited to nucleoside reverse transcriptase inhibitors (NTRIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), proteaseinhibitors (PIs), fusion and entry inhibitors, pharmacokinetic enhancesand integrase strand transfer inhibitors (INSTIs).

Example 7—Treatment of Germinal Center Lymphomas, for Example BurkittsLymphoma

A patient with a germinal center lymphoma such as but not limited toBurkitt's Lymphoma or diffuse large B-cell lymphoma (DLBCL) is treatedwith glucocorticoid immune suppressant, or with dexamethasone. Acutehigh dose dexamethasone (as base) doses range from about 15 mg/kg toabout 26 mg/kg, with doses between about 15 mg/kg and about 21 mg/kgbeing preferred. The treatment eliminates the niches in the spleen wherethe germinal center lymphomas bind and sends the cells into thecirculation where they can be eliminated more completely, or with lowerdoses, of standard chemotherapy such as R-CHOP, or by antibodies to CD20such as Rituxan, Bexxar, or Zevalin, or by antibodies to CD22 or CD70such as Lymphocide or Vorsetuzumab mafodotin, or by Bcl-2 inhibitorssuch as Oblimersen sodium, ABT-737 (oral form navitoclax, ABT-263), orFenretinide, or by Syk inhibitors such as Fostamatinib or Tamatinib, orby proteasome inhibitors such as Bortezomib (Velcade), or COMPADME,CODOX-M/IVAC. Relapse rates are reduced and disease free survival ratesare increased.

Example 8—Conversion of a Dexamethasone Dose to an Equivalent Dose ofAnother Glucocorticoid

To calculate the equivalent dosing for another glucocorticoid, the doseof dexamethasone is entered into a publicly available glucocorticoidconversion calculator, preferably http://www.medcalc.com. Then the totaldosing is determined based on the half-life of the glucocorticoid. Forinstance, 3 to 12 mg/kg dexamethasone converts to 19 to 75 mg/kgprednisone. Since prednisone's biologic half-life is about 20 hours,while dexamethasone's biologic half-life is about 36 to 54 hours.Therefore, prednisone would be dosed between 19 to 75 mg/kg every 24hours for equivalent biologic dosing.

Example 9—Treatment of Patients with Autoimmune Diseases with Prednisone

A patient with an autoimmune disease such as, but not limited to: SLE,psoriasis, rheumatoid arthritis, sporiatic arthritis, type I diabetes,multiple sclerosis, Sjogren's Syndrome, scleroderma, Grave's Disease,Hashimoto's thyroiditis, Celiac Disease, Addison's Disease, MyastheniaGravis, Autoimmune hepatitis, Antiphospholipid syndrome, biliarycholangitis, can be treated with acute high dose prednisone. Acute highdose prednisone doses range from about 19 mg/kg to about 150 mg/kg, withdoses between about 56 mg/kg and about 112 mg/kg being preferred, with arepeat (second) administration of this dose 24 hours later and anoptional repeat (third) administration of this dose 48-72 hours afterthe initial dose.

B lymphocyte numbers are reduced by greater than 90% with acute highdose prednisone doses, and as memory B cells make up approximately 50%of the B cell compartment in people over age 20, memory B cellpopulations are also reduced by greater than 90%. The patient'sautoimmune attacking B cells have apoptosed and the patient ceases tohave active self-immune attacks. The patient's physical symptoms areimproved or eliminated. Remission from the autoimmune disease lastsindefinitely in most patients, however, should the patient relapse thena repeat dose of the prednisone can be administered. Repeat treatmentscan occur as often as once per month if necessary, but preferably notmore than one a year, and most preferably not more than once every 5years.

Example 10—Immunosuppressant Lymphodepletion in Female Mice 24-48 Hoursafter Acute Administration of Dexamethasone, with Neutrophil, RBC,Platelet and Stem Cell Sparing Properties

Preliminary dose escalation studies performed in naïve mouse and ratmodels showed that administration of high-dose dexamethasone results incomplete lymphodepletion. High-doses of dexamethasone were able toinduce ˜98% reduction in CD4+, CD8+, Tregs and B cells populationmeasured 48 hours after administration, supporting rapid ablation ofautoimmune pathophysiologic substrates. Early stage validation showedthat acute high dose dexamethasone has 2-3 hour half-life bypharmacokinetic and a pharmacodynamics half-life of 4-5 days, whichexclude prolonged immune suppression. In addition, oral dosing of acutehigh dose dexamethasone has comparable effects to IV dosing, whichsupports the use of acute high dose dexamethasone as a single oraltreatment.

Oral acute administration of dexamethasone to C57B1 female mice at HEDof 12 mg/kg, HED 15 mg/kg, 18 mg/kg demonstrated that female mice wereless sensitive to dexamethasone base with lymphoablation not completelyevident until doses above 18 mg/kg.

Acute high dose dexamethasone activates receptor-mediated apoptosis viathe caspase pathway and lympho-depletes or lympho-ablates alllymphocytes depending on the dose used. As expected from itsreceptor-mediated mode of action, dexamethasone induces lymphodepletionsparing neutrophils, platelets, and Red Blood Cells (RBCs) due to thelack of or due to different glucocorticoid receptors on these cells. Atendency to elevate neutrophil counts above placebo in both peripheralblood and bone marrow was observed with high doses of dexamethasone,supporting possible protection against infections during lymphodepletiontreatments.

The non-myeloablative regimen represented by acute high dosedexamethasone could, therefore, eliminate the need for transfusions ofstem cells for hematopoietic recovery after immune-reset.

REFERENCES

A number of publications are cited above in order to more fully describeand disclose the invention and the state of the art to which theinvention pertains. Full citations for these references are providedbelow:

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The invention claimed is:
 1. A method of treating a lymphocyte mediateddisease in a subject, comprising administering a pharmaceuticalcomposition comprising dexamethasone or betamethasone and one or morepharmaceutically acceptable carriers, preservatives, and/or chelatingagents, to the subject at a dose of about 9 to about 26 mg/kg humanequivalent dose (HED) of dexamethasone base, wherein administering thepharmaceutical composition depletes splenic, peripheral blood, thymicand bone marrow lymphocytes in the subject by at least 50%; wherein themethod does not comprise co-administration of a chemotherapy agent; andwherein the lymphocyte mediated disease is not graft versus host disease(GVHD).
 2. The method of claim 1, wherein the lymphocyte mediateddisease is an autoimmune disease.
 3. The method of claim 1, wherein thelymphocyte mediated disease is residual HIV disease.
 4. The method ofclaim 1, wherein the lymphocyte mediated disease is a germinal centerlymphoma.
 5. The method of claim 1, wherein the lymphocyte mediateddisease is an allergic disorder.
 6. The method of claim 2, wherein theautoimmune disease is selected from Type 1 diabetes, multiple sclerosis,amyotrophic lateral sclerosis, scleroderma, pemphigus, and lupus.
 7. Themethod of claim 1, wherein the pharmaceutical composition comprises apreservative, wherein the preservative is a sulfite.
 8. The method ofclaim 1, wherein the pharmaceutical composition comprises a chelatingagent, wherein the chelating agent is EDTA.
 9. The method of claim 1,wherein the glucocorticoid is dexamethasone, wherein the dexamethasoneis selected from dexamethasone base, dexamethasone sodium phosphate anddexamethasone acetate.
 10. The method of claim 9, wherein thedexamethasone is dexamethasone sodium phosphate.
 11. The method of claim1, wherein the pharmaceutical composition is administered as a singleacute dose or a total dose given over about a 72 hour period.
 12. Themethod of claim 1, wherein the pharmaceutical composition isadministered as an intravenous (IV) or oral dose.
 13. The method ofclaim 1, wherein the pharmaceutical composition is an aqueous solution.14. The method of claim 1, wherein the pharmaceutical composition isadministered at a dose of at least about 9 mg/kg, at least about 10mg/kg, at least about 11 mg/kg, at least about 12 mg/kg, at least about15 mg/kg, at least about 18 mg/kg, or at least about 24 mg/kg of a humanequivalent dose (HED) of dexamethasone base.